Lipopeptide Antibiotics - an overview

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Permeabilizing Antibiotics: Lipopeptide Antibiotics and Lantibiotics

Daptomycin (produced by Streptomyces roseosporus) and polymyxin B (derived from Bacillus polymyxa) (Figure 2) are both lipopeptide antibiotics that comprise a fatty acid chain and a charged cyclic peptide and are in clinical use. Daptomycin and polymyxin B have different antibiotic spectra and are effective against Gram-positive and Gram-negative bacteria, respectively. The mechanisms of these two drugs are similar; the fatty acid chain is used to bind to the lipophilic membrane, and the peptide component is responsible for depolarizing the membrane, making it more permeable. For example, the positively charged polymyxin B (due to the presence of multiple amino groups, see Figure 2) cannot penetrate the thick peptidoglycan layer of Gram-positive bacteria. It is however able to bind to lipid A, which is found on the outer membrane in Gram-negative bacteria. The binding of polymyxin to the outer membrane causes alteration to the membrane structure, leading to enhanced permeability and depolarization.

Overall, daptomycin is negatively charged (due to the presence of multiple carboxyl groups, see Figure 2) and does not penetrate the outer-membrane of Gram-negative bacteria. Daptomycin is however active against Gram-positive bacteria and appears to bind the cell membrane of Gram-positive bacteria to cause depolarization. Various resistant mechanisms of lipopeptides have been proposed including adaptive response, secretion of degradation enzymes, and alteration of the membrane structure (Moore et al., 1984), yet detailed mechanistic studies for the resistance are lacking. A study in 2006 investigated bacterial resistance to various drug classes, using 480 soil-derived bacterial isolates (D'Costa et al., 2006). Interestingly, all 480 isolates displayed high levels of resistance to Daptomycin. Detailed analysis of these resistant bacteria could probably shed more light on how bacteria develop resistance to lipopeptide antibacterials.

Lantibiotics are a family of cyclic peptides with thioether bond and unnatural amino acids (e.g., dehydroalanine and 2-aminoisobutyric acid). One of the most studied compounds among lantibiotics is nisin (Figure 13). Nisin (produced by Lactococcus lactis) comprises of 34 amino acid residues and five thioether rings. Nisin disrupts cell function by promoting the formation of pores in the cell membrane (Figure 14). Nisin does not penetrate the lipid-rich outer membrane of Gram-negative bacteria or mycobacteria and therefore it is only effective against Gram-positive bacteria. It has been used as food preservative for decades.

Figure 13. Structure of nisin.

Figure 14. (a) Structure of lipid II. (b) Mechanism of action of nisin. Nisin binds to lipid II with its cargo, glycopeptide, and forms a pore in the cytoplasmic membrane.

Figure 14(b) is adapted from Brötz, H., Sahl, H.G., 2000. New insights into the mechanism of action of lantibiotics – diverse biological effects by binding to the same molecular target. J. Antimicrob. Chemother. 46, 1–6.The permission is granted by Oxford University Press.

Nisin is able to disrupt the bacterial membrane after interacting with lipid II. Lipid II, a polyprenyl derivative as shown in Figure 14(a), plays an important role in the biosynthesis of the peptidoglycan. The precursor of the peptidoglycan (a glycopeptide) is produced in the cytosol and needs to be transported across the cell membrane. The hydrophobic isoprenyl chain of lipid II anchors into the cell membrane, and then flips over its cargo, the glycopeptides precursor for the peptidoglycan, into the exterior of the cell membrane for further processing. Nisin forms a tight 1:1 complex with lipid II. The pyrophosphate unit of lipid II fits nicely into a cavity created by the N-terminus of nisin. Interestingly, the dominant interaction is via hydrogen bonds of the backbone NH (and not via side-chain interactions) of nisin to the pyrophosphate unit. Following the initial complex formation between nisin and lipid II, nisin forms higher-order structures leading to pore formation in the cell membrane (Figure 14(b)). Interestingly, some lantibiotics that also contain similar A/B rings of nisin do not form pores but are also potent antibiotics (Hasper et al., 2006). Therefore, binding to lipid II alone is not sufficient for pore formation. Plausibly, these antibiotics that contain the A/B rings, which are found in nisin, bind to lipid II and prevent it from performing its key function in peptidoglycan synthesis.

Although nisin is not used clinically because of pharmacokinetic issues, such as poor bioavailability and fast metabolism, various lantibiotics, which have better pharmacokinetic properties, have entered into clinical trials (Willey and van der Donk, 2007). Lipid II plays a key role in peptidoglycan synthesis so drugs that inhibit the biosynthesis of lipid II also have the potential to become antibacterial agents are certainly worth pursuing.

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4.1 Introduction

Within the vast structural diversity of antimicrobial peptides, lipopeptides are of special importance highlighted by daptomycin and the polymyxins, which are valuable commercial antibiotics of therapeutic use.

Lipopeptides are characterized by the existence of a mostly macrocyclic peptidic core to which a hydrocarbon lipid tail (usually > C6) is linked via the N-terminus and may include hydroxy groups and unsaturation(s). The lipid tail interacts with lipids of bacterial and potentially mammalian cell membranes generally enhancing the bactericidal effect of lipopeptide antibiotics. The macrocyclic peptidic core is important for specific interactions of the lipopeptide with lipid and/or protein targets. Nevertheless, the lipophilic hydrocarbon tail does not generally improve desirable drug properties like good aqueous solubility and is prone to generate unspecific binding to phospholipid bilayers, thus increasing potential off-target effects.

In the last decades, a large number of antimicrobial lipopeptides of natural source has been discovered. Besides the mentioned daptomycin and polymyxins, there are many additional interesting lipopeptides, for example, the actinocarbasin (arylomycin D; 19, Figure 4), globomycin, enopeptins, or friulimicin (11,52).

Within this part of the review, we would like to focus only on a few selected examples of lipopeptide antibiotics that stand out based on their novel MoA, have available in vivo efficacy data, and/or moved into preclinical or clinical development.

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2.19.2.1.3 Lipopeptide antibacterials

That directly searching for novel antibiotics from microbes is still a viable method in certain cases can be seen from the data on daptomycin (19, Figure 3), which, although having a somewhat chequered career in moving from Lilly to Cubist, was developed by Cubist and approved in 2003 by the Food and Drug Administration (FDA) as a Gram-positive active antibiotic now known to act on potassium channels. By use of modern genetic techniques coupled to chemistry, it is now possible to produce biosynthetically modified scaffolds (the so-called ‘nonnatural natural products’) that widen the chemical universe for antibiotic discovery. Using such techniques, Baltz’ group at Cubist have recently published their results, which included the production of one variant that had activity against an Escherichia coli strain with an outer membrane modification that rendered it more permeable.29 This paper should be read in conjunction with their earlier review in 2005 covering both daptomycin and earlier lipopeptides.30

Figure 3. Lipopetide antibacterials.

Another rather old antibiotic that is still being optimized for use is ramoplanin, which as currently utilized is a lipopeptide antibiotic complex, consisting of small amounts of factors A1, A′1, A′2, A3, and A′331,32 and factor A2 (‘ramoplanin’) (20, Figure 3) isolated from Actinoplanes sp. ATCC33076. Factor A2 is the major component of the complex and is being evaluated in phase III trials by Oscient Pharmaceuticals for the treatment of Clostridium difficile-associated diarrhoea (CDAD).33,34 Ramoplanin (20, Figure 3) exerts its antibacterial activity by binding to the peptidoglycan intermediate Lipid II (C35-MurNAc-peptide-GlcNAc) rather than to the vancomycin tripeptide, thus disrupting bacterial cell wall synthesis.32,35,36 The compound has received orphan drug status in the EU for the treatment of vancomycin-resistant Enterococci (VRE) but no trials are yet reported.

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4 Lipopeptides

4.1 Introduction

Within the vast structural diversity of antimicrobial peptides, lipopeptides are of special importance highlighted by daptomycin and the polymyxins, which are valuable commercial antibiotics of therapeutic use.

Lipopeptides are characterized by the existence of a mostly macrocyclic peptidic core to which a hydrocarbon lipid tail (usually > C6) is linked via the N-terminus and may include hydroxy groups and unsaturation(s). The lipid tail interacts with lipids of bacterial and potentially mammalian cell membranes generally enhancing the bactericidal effect of lipopeptide antibiotics. The macrocyclic peptidic core is important for specific interactions of the lipopeptide with lipid and/or protein targets. Nevertheless, the lipophilic hydrocarbon tail does not generally improve desirable drug properties like good aqueous solubility and is prone to generate unspecific binding to phospholipid bilayers, thus increasing potential off-target effects.

In the last decades, a large number of antimicrobial lipopeptides of natural source has been discovered. Besides the mentioned daptomycin and polymyxins, there are many additional interesting lipopeptides, for example, the actinocarbasin (arylomycin D; 19, Figure 4), globomycin, enopeptins, or friulimicin (11,52).

Within this part of the review, we would like to focus only on a few selected examples of lipopeptide antibiotics that stand out based on their novel MoA, have available in vivo efficacy data, and/or moved into preclinical or clinical development.

4.2 Tridecaptins

Tridecaptins (Figure 2) are nonribosomally synthesized natural products within the class of linear cationic tridecapeptides, which have been first isolated in 1978. Tridecaptins display good antimicrobial activity against Gram-negative bacteria with single-digit MICs [μg/mL] on Enterobacteriaceae and weaker activity against A. baumannii and moderate activity against P. aeruginosa (53,54). The antimicrobial activity against Gram-positive bacteria is generally moderate. SAR and MoA studies of one member of this family, Tridecaptin A1 (TriA1; 5, Figure 2) isolated from Paenibacillus terrae, have been described in more detail (54–56). Cochrane et al. have observed that the structure of the lipid tail of TriA1 could be varied without loss of antimicrobial activity (55). The analogue OctTriA1 (6) containing an octanoyl lipid chain fully retained its activity against all organisms (55). In a first step, TriA1, and also the derivatives Ent-TriA1 (enantiomer), Oct-TriA1, and H-TriA1 (unacylated TriA1) bind with a similar affinity to lipopolysaccharide located in the outer membrane of Gram-negative bacteria (54). However, the unacylated H-TriA1 (4) as well as analogues with shorter lipid tail (<C6) are significantly less active, pointing to the important contribution of the lipid chain to the antimicrobial activity by interaction with phospholipids. TriA1 is a membrane-targeting peptide but does not act by generic membrane lysis mechanism like many other AMPs. The enantiomer of TriA1 (Ent-TriA1) is fourfold less active than the natural peptide, suggesting that TriA1 interacts with a chiral target. Indeed, TriA1 binds to lipid II, the monomeric intermediate in the peptidoglycan biosynthesis, located on the inner surface of the plasma membrane. Interestingly, it could be shown that TriA1 binds much weaker to the lipid II of Gram-positive bacteria (structure cf. Figure 9), which explains the lower activity on Gram-positive bacteria. In line with the postulated mechanism, Cochrane et al. nicely elucidated by ITC binding studies that the enantiomer of TriA1 (Ent-TriA1) lacks binding to lipid II (54). In addition to the interaction with lipid II, disruption of the proton motive force, a vital process to produce bacterial ATP within the bacterial membrane, seems to be affected and could explain the antibacterial activity of TriA1 (54). Another member of the tridecaptin class, tridecaptin M (7), exhibited good in vivo efficacy at 10 mg/kg bid s.c. in a neutropenic mouse thigh infection model against a colistin-resistant strain of K. pneumonia (57). Although reported to be not hemolytic on rabbit erythrocytes at 100 μg/mL, hemolysis of 50% of cells at 200 μg/mL has been observed (57). Therefore, possible future lead optimization based on the tridecaptin scaffold should focus on minimizing hemolysis and cytotoxicity (58). The design of synergistic dual-acting bifunctional antimicrobial conjugates (chimeric antibiotics) has become a popular approach to increase antimicrobial activity (59). Vederas and colleagues synthesized antibiotics conjugates starting from H-TriA1 (unacylated TriA1) linked via copper(I)-mediated click chemistry to vancomycin, rifampicin, and erythromycin analogues (60). These chimeric compounds showed enhanced but not synergistic activity in vitro in checkerboard studies. In vivo, survival rates were compared to both parent drugs in combination and alone in a K. pneumonia murine survival experiment, all conjugates and both parent compounds being administered intravenously (tail vein). Only the conjugate H-triA1-erythromycin led to a 2-fold higher survival rate compared to both parent drugs in combination (60). However, surprisingly, in this study, vancomycin alone, which has no activity on Gram-negative bacteria, showed an 80% survival rate in this model.

Figure 2. Structures of Tridecaptins; structures of Malacidin A and B. Amino acids of tridecaptin M (7) are annotated in brackets.

4.3 Malacidins

As mentioned before, it is assumed that less than 1% of all environmental microbes have been cultured in laboratory settings as a source to discover new antimicrobial molecules (4). Through applying emerging technologies such as environment-mimicking cultivation and genome mining of Biosynthetic Gene Clusters (BGCs), Brady and colleagues have discovered malacidin A and B (8a, 8b; Figure 2) as members of a new class of calcium-dependent macrocyclic lipopeptide antibiotics (61,62). Malacidin A consists of a macrocyclic nonapeptide, containing four nonproteinogenic amino acids, and an unsaturated C9-fatty chain acylated to an exocyclic β-methylaspartic acid. The total synthesis of malacidin A has been reported by Sun et al. and the absolute configurations of its five nonproteinogenic amino acid residues have been elucidated (63). Malacidin A exhibits broad activity against Gram-positive bacteria including methicillin-resistant S. aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) and has potent antimicrobial activity (MIC range 0.2–2 μg/mL) in the presence of Ca2+ ions. Malacidin A was also able to clear S. aureus infection in a skin infection rat model. The MoA of malacidins involves binding to lipid II as described for other known antibiotics like vancomycin and teixobactin. However, since no cross resistance to vancomycin has been observed, binding of malacidin A to lipid II must be different from the binding mode of vancomycin.

4.4 Lipopeptides of the polymyxin/colistin/octapeptin family

The polymyxin lipopeptide antibiotic family was discovered in 1947 and is characterized by its potent, specific activity against Gram-negative bacteria (64). Polymyxin B (Polymyxin B1, 9, Figure 3), polymyxin E (colistin, 10), and colistin methanesulfonate (CMS, 11) were used clinically. However, they were subsequently gradually withdrawn from clinical practice in the 1960s after several reports of partially severe nephrotoxicity and rather mild neurological effects in a large number of patients (65,66). CMS (11) is a colistin prodrug that is applied parenterally and by inhalation (67). With the urgent need of new antibiotics due to increased emergence of multidrug-resistant bacteria and new and more stringent dosing regimens (68), colistin (10) has experienced a revitalization in the clinical use as a “last resort” antibiotic against P. aeruginosa, A. baumannii, and K. pneumoniae (69). Due to conservative use of polymyxins in the last century, resistance development has been rare and mostly based on modifications of lipid A (70). However, with their late use as last-line treatment against MDR infections and for their massive overuse in agriculture and poultry, resistance in clinical pathogens is on a global rise, especially the worrisome plasmid-borne mcr containing strains (71,72).

Figure 3. Structures of the polymyxin/colistin and octapeptin antibiotics.

The mode of action of these rapidly bactericidal antibiotics is still not completely understood. Presumably, in an initial step, the positively charged polymyxins bind to the negatively charged lipid A part of lipopolysaccharides (LPS) followed by displacement of Ca2+ ions (73). The peptides cross the outer membrane through a “self-promoted uptake” mechanism and then interact with the cytoplasmic membrane to inhibit cellular energization, and possibly cause inhibition of cell division and/or cytoplasmic membrane permeabilization and subsequent cell death (74). In this context, the lipid tail of the polymyxins is crucial for activity as a shortened variant of polymyxin without the hydrophobic N-terminal fatty-acyl chain exhibits much reduced activity (75). In the context of the revival of their use, further research in their mechanism of resistance and action led to the proposal of new alternative mechanisms. In particular, research groups have explored the ability of polymyxins to bind to ribosomes, prevent cell division, and inhibit bacterial respiration (74).

The high need for new antibiotics in particular against Gram-negative bacteria (last new class were the Quinolones over 50 years ago (21)) and probably also the relative straightforward chemical accessibility has spurred the search for novel synthetic polymyxin derivatives and more recently also for the closely related octapeptin analogues (12, Figure 3) (64), with the potential to overcome polymyxin resistances and reduced renal toxicity (76).

Modifications within the cyclic heptapeptide ring, variation of the exocyclic amino acids and with particular focus, the replacement of the fatty-acyl chain lead to equally potent derivatives compared to the polymyxins B and E and with some derivatives partially overcoming polymyxin resistance. Many of these next-generation polymyxins have been extensively reviewed elsewhere (76,77). However, designing and optimizing compounds with reduced renal toxicity in animal settings translating reliably into human is still a challenge. So far, none of the new active analogues have shown improvement of the therapeutic index compared to polymyxins particularly regarding nephrotoxicity.

Recently, Brown et al. have described an interesting approach to tackle the problem of nephrotoxicity (76). SPR206 (14; Figure 3) is a polymyxin B analogue where the lipid tail was replaced by (S)-4-amino-3-(3-chlorophenyl)butanoic acid. SPR206 has been selected as a result of an intriguing medicinal chemistry effort based on the understanding of structure–activity relationship of antimicrobial activity, in vitro cytotoxicity against human kidney proximal tubular epithelial cell line (HK-2), and kidney exposure (78). The exact mechanism of renal toxicity of polymyxins is not completely understood, but it has been shown that polymyxins are reabsorbed through the proximal tubular cells and accumulate to exert renal toxicity (79). SPR206 showed 12 times lower cytotoxicity toward the HK-2 cell line compared to polymyxin B1 resulting in lower nephrotoxicity in an acute (24 hours) mouse in vivo model based on biomarkers and kidney histopathology assessment at comparable dose. Interestingly, kidney exposure of SPR206 in mice was similar compared to polymyxin B1. Recently, SPR206 has finished phase-I trial (NCT037992308). It will be interesting to see if SPR206 can successfully further progress in the clinic.

In the search of polymyxin derivatives with more favorable antimicrobial activity on colistin-resistant strains and nephrotoxicity profiles, derivatives of octapeptins (12, e.g., octapeptin C4 and octapeptin B5 (13; Figure 3) have been investigated (80,81). Battacin (13) shows interesting antimicrobial activity (MICs = 2–4 μg/mL) against some multidrug-resistant clinical strains of E. coli and P. aeruginosa (64). The overall positive charge together with the N-terminal fatty-acyl chain was considered to be a main contributor to the renal toxicity within the polymyxin class (82). Interestingly, octapeptins contain at least one cation less compared to polymyxins. However, octapeptin C4 shows a fourfold higher toxicity compared to polymyxin B on HK-2 cell line (80). Recently, a group at the University of Queensland, Australia, has received significant funding to further develop new octapeptin derivatives (83).

Different approaches were explored by Vaara and coworkers (84). By reducing the number of positive charges and altering of the exocyclic amino acids of polymyxin B, they obtained potentially less nephrotoxic derivatives. Polymyxin nonapeptide (PMBN) is a polymyxin derivative lacking the N-terminal fatty-acyl chain and the last exocyclic Dab-residue. It has no antibiotic activity; however, it binds to LPS, permeabilizes the outer membrane of Gram-negative bacteria, and thereby potentiates synergistically the antimicrobial activity of antibiotics like rifampin, clarithromycin, and azithromycin when applied in combination. NAB741 (SPR741) is a PMBN-derivative with three positive charges (at physiological conditions) and has completed two phase-I trials envisaged to be used as potentiator in combination with other antibiotics. In future clinical studies, SPR741 will be combined with other antibiotics using the i.v. route to show hopefully a clinical benefit (6).

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Biosynthesis of antibacterial nucleoside antibiotics: pacidamycin, liposidomycin, tunicamycin, and capuramycin

This group, which is also referred to as uridine-based nucleosides, can be further divided into four subfamilies: uridyl peptide antibiotics, uridyl lipopeptide antibiotics, uridyl lipodisaccharide antibiotics, and uridyl glycosylpeptide antibiotics. The uridyl peptide antibiotics encompass pacidamycin, napsamycin, mureidomycin, and sansanmycin. They share a common structural scaffold, a unique 3′-deoxy-4′,5′-enamino-uridine nucleoside linked to a pseudotetra- or pentapeptide backbone (Figure 1), and exhibit selective antibacterial activity against Pseudomonas aeruginosa, a common nosocomial pathogen that is intrinsically resistant to various clinically used antibiotics. Sansanmycin also displays inhibition against multidrug-resistant Mycobacterium tuberculosis strains [4]. The uridyl lipopeptide antibiotics are represented by liposidomycin, caprazamycin, muraymycin, muraminomicin, and A-90289. They are structurally characterized by a 5′-C-glycyluridine (GlyU), an aminoribofuranoside, a diazepanone, and variable fatty acid side chains (Figure 1). The uridyl lipodisaccharide antibiotics are represented by tunicamycins comprising an unusual 11-carbon aminodialdose core, uracil, N-acetylglucosamine (GlcNAc), and variable fatty acyl moieties (Figure 1). The uridyl glycosylpeptide antibiotics include capuramycin and related compounds (A-503083, A-102395, and A-500359), which are characterized by a 5′-carbamoyluridine (CarU), an unsaturated hexuronic acid, and an aminocaprolactam ring (Figure 1).

Figure 1. Chemical structures of representative nucleoside antibiotics. Parts shaded in pink indicate the nucleoside moieties. CarU, 5′-carbamoyluridine; DABA, 2,3-diaminobutyric acid; GlyU, 5′-C-glycyluridine; PABA, p-aminobenzoic acid.

Comparison of gene clusters shows high sequence similarities of the corresponding proteins involved in the biosynthesis of pacidamycin, napsamycin/mureidomycin, and sansanmycin [5–8]. Most genetic and biochemical evidence comes from pacidamycin biosynthesis. Biosynthesis of the peptide backbone has been investigated extensively by Christopher Walsh and colleagues. Apart from the two non-proteinogenic amino acids meta-tyrosine (m-Tyr) and 2,3-diaminobutyric acid (DABA), other amino acids are thought to originate from primary metabolism. DABA is postulated to be synthesized from L-threonine and ammonia [6]. m-Tyr is generated from L-phenylalanine by a novel iron (II)-dependent phenylalanine-3-hydroxylase (PacX) [9]. These amino acids are linked by nine proteins that constitute the non-ribosomal peptide synthetase (NRPS) assembly line [10]. One exceptional feature of this assembly is that it does not start at the N-terminal residue AA1 and proceed to the C-terminal residue AA5. Instead, assembly is initiated by the activation and methylation of the core residue DABA and the chain is then built from the middle outward in both directions [11]. It should be noted that the transferase PacB is responsible for the transfer of the alanyl residue from alanyl-tRNA to the N terminus of the tetrapeptide intermediate yielding the pentapeptide scaffold [12]. This enzyme is unusual compared with typical NRPSs in that it catalyzes peptide bond formation in a tRNA-dependent way, hijacking an aminoacyl-tRNA from the primary metabolic pathway to the secondary antibiotic biosynthetic pathway. Rebecca Goss and colleagues have shown that the biogenesis of the unique 3′-deoxy-4′,5′-enamino-uridine proceeds through three steps (Figure 2A). Uridine is first oxidized by flavin-dependent dehydrogenase (Pac11/PacK) to uridine-5′-aldehyde (UA), which is then subjected to 3′,4′-dehydration and 5′-transamination through the action of a Cupin family enzyme (Pac13/PacM) and pyridoxal-5′-phosphate (PLP)-dependent aminotransferase (Pac5/PacE) [13]. The free-standing condensation protein PacI is thought to then catalyze the release of the assembled peptide and its linkage with the nucleoside scaffold to build pacidamycins [10].

Figure 2. Representative pathways for biosynthesis of nucleoside antibiotics. (A) Biosynthesis of nucleoside skeleton from uridine in uridyl peptide antibiotics. (B) Biosynthesis of aminoribosyl moiety from uridine-5′-monophosphate (UMP) in lipopeptide antibiotics. (C) The tandem reactions catalyzed by CapS and CapW in the amide bond formation of A-503083 B. (D) Biosynthetic pathway for the formation of cytosylglucuronic acid (CGA) and hydroxymethyl-CGA (HM-CGA) from cytidine-5′-monophosphate (CMP) in cytosine-derived nucleoside antibitics. HM-CMP, hydroxymethyl-CMP; NDP, nucleoside diphosphate; NTP, nucleoside triphosphate; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; UA, uridine-5′-aldehyde. Adapted from [13,15,20,35].

Six genes (lipK, lipL, lipO, lipP, lipM, and lipN) are essential for the formation of the aminoribosyl moiety in lipopeptide antibiotics (Figure 2B). The pathway is initiated by oxidative dephosphorylation of uridine-5′-monophosphate (UMP) to UA, catalyzed by a non-heme iron (II)-dependent dioxygenase (LipL) [14]. This is in contrast to the first step in the biosynthesis of the pacidamycin nucleoside (3′-deoxy-4′,5′-enamino-uridine), where uridine is oxidized to UA. Subsequently, UA is converted to 5′-amino-5′-deoxyuridine through the action of the L-methionine:UA aminotransferase (LipO). 5′-Amino-5′-deoxyuridine then serves as the substrate for a phosphorylase (LipP) to generate 5′-amino-5′-deoxy-α-D-ribose-1-phosphate. This sugar-1-phosphate is then processed by two enzymes in a manner that parallels typical glycosylation events: the 5-amino-5-deoxy-α-D-ribose-1-phosphate is activated by a nucleotidylyltransferase (LipM) as the nucleotide-5′-diphosphate (NDP)-sugar [uridine-5′-diphosphate (UDP)-5′-amino-5′-deoxy-α-D-ribose] and the sugar is transferred by a glycosyltransferase (LipN) to an acceptor molecule to generate the final disaccharide [15]. The potential acceptor for UDP-5-amino-5-deoxy-α-D-ribose is 5′-C-glycyluridine, which is generated by a PLP-dependent L-threonine:uridine-5′-aldehyde transaldolase, LipK [16]. The rest of the biosynthetic steps, including the formation of the diazepanone ring and attachment of the fatty acid, remain elusive.

The minimal gene cluster for tunicamycin biosynthesis has been identified [17,18] and TunA and TunF were characterized respectively as a dehydratase and an epimerase, participating in the formation of a unique enol ether intermediate [UDP-6-deoxy-5,6-ene-N-acetylgalactosamine (GalNAc)] from UDP-GlcNAc and uridine [19]. A deeper understanding of the rest of the enzymatic processes may offer the opportunity to alter this unique natural product scaffold and reduce its cytotoxicity to mammalian cells. The biosynthetic gene clusters for A-500359s and A-503083s have been reported [20,21]. CapP is an ATP-dependent capuramycin phosphotransferase that transfers the γ-phosphate of ATP to the 3′-hydroxyl of the unsaturated hexuronic acid moiety of A-503083 B and confers selective resistance to A-500359 B [22]. CapS functions as an S-adenosylmethionine (SAM)-dependent carboxyl methyltransferase and activates the carboxylic acid substrate to the methyl ester at the expense of SAM. In the presence of free L-aminocaprolactam, CapW converts the methyl ester to A-503083 B by generating a new amide bond (Figure 2C). The tandem reactions catalyzed by CapS and CapW represent an ATP-independent strategy for carboxylic acid activation and intermolecular amide bond formation [20], a departure from the widespread mechanism of amide bond formation that requires the hydrolysis of ATP [23].

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Lipopeptides (Daptomycin)

Daptomycin is a cyclic lipopeptide antibiotic with a good soft tissue penetration. It has activity against most gram-positive bacteria including MRSA and VRE, but no activity against gram-negative microorganisms.92 Therefore, with the exception of microbiologically-proven infectious complications with gram-positive microorganisms, Daptomycin should be used as part of a combination therapy for patients with postoperative complications after pancreatic surgery.

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Daptomycin

This lipopeptide antibiotic was approved by the FDA in 2003 for the treatment of complicated skin and soft tissue infections, including those with vancomycin-susceptible E. faecalis, and it was granted an additional indication in 2006 for S. aureus (including MRSA) bacteremia and right-sided endocarditis. Daptomycin is not FDA-approved for the treatment of E. faecium (regardless of susceptibility) or for VRE infections. The molecular events involved in its antibacterial action have not yet been completely elucidated, and it is thought that daptomycin inserts into the bacterial cell membrane in a calcium-dependent manner with preferential binding to septal areas, producing alterations of membrane homeostasis and cell division. These effects eventually lead to bacterial cell death by an unknown mechanism. In vitro, daptomycin exhibits rapid concentration-dependent bactericidal killing of enterococci and, in vivo, the area under the curve (AUC; free drug)/MIC ratio appears to be the best parameter that correlates with clinical success.113 The activity of daptomycin in vivo appears to be affected by its high binding affinity to albumin (90% to 94%), and the unbound fraction might be crucial in the treatment of endovascular enterococcal infections when bactericidal therapy is a requirement. This notion, plus the higher MICs and breakpoints than for staphylococci, has led to the notion that higher doses (8 to 12 mg/kg) may be of clinical benefit (see Figs. 202-2 and 202-3). Combination therapies that include daptomycin have been used in recalcitrant enterococal infections. For example, failure of daptomycin monotherapy in the treatment of E. faecium endocarditis was overcome by the use of a combination of high-dose daptomycin (8 mg/kg/day), high-dose ampicillin, and gentamicin.114 Similarly, the combination of daptomycin, gentamicin, and rifampin successfully treated a case of prosthetic valve endocarditis caused by a vancomycin-resistant isolate of E. faecium that failed with linezolid,115 and in vivo experiments have demonstrated that the renal toxicity of gentamicin appears to be attenuated by the concomitant use of daptomycin. Combinations of daptomycin plus ampicillin or ceftaroline have been successfully used to treat recurrent deep-seated enterococcal infections that have failed daptomycin monotherapy.115a,115b An additional anecdotal successful combination used in the treatment of endocarditis caused by a multidrug-resistant (including HLR to aminoglycosides) E. faecium included the combination of daptomycin (6 mg/kg every 48 hours) and tigecycline.116 Enterococci resistant to daptomycin have been well documented (daptomycin MICs range from 6 to >32 mg/L), although they still remain relatively rare. Daptomycin resistance emerging during prolonged therapy (average exposure to daptomycin of 18 days) has been documented in E. faecalis, E. faecium, and E. durans isolates, although it has also been observed in isolates without previous exposure to the antibiotic.117 All patients had a certain degree of immunosuppression and were treated for bacteremia and/or endocarditis. Recent data suggest that a pivotal event in the mechanism of daptomycin resistance in enterococci involves changes in genes predicted to orchestrate the bacterial cell envelope stress response (designated liaFSR) and encoding enzymes of phospholipid metabolism (e.g., cardiolipin synthase).118 Moreover, mutations in liaFSR in daptomycin-susceptible enterococci have been shown to lead to in vitro daptomycin tolerance and abolish the bactericidal activity of the antibiotic against these organisms.119

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URL: https://www.sciencedirect.com/science/article/pii/B9781455748013002022

Daptomycin

Daptomycin (DAP) is a lipopeptide antibiotic with in vitro bactericidal activity against both E faecalis and E faecium, including VRE.61 DAP consists of a cyclic polypeptide core with a lipid tail that facilitates insertion into the bacterial membrane in a calcium-dependent manner, a characteristic shared with cationic peptides of the innate immune response.62 The precise mechanism by which the antibiotic mediates cell death is not known, but DAP-treated bacteria rapidly lose membrane physicochemical properties with disruption of the ionic gradient used to drive many biosynthetic pathways.63 It is postulated that DAP forms a multimeric pore structure in the presence of phosphatidylglycerol, with 2 tetramers aligned opposite each other on the inner and outer leaflet of the membrane.64 Enrichment of the membrane with cardiolipin (CL), with its 4 bulky fatty acyl chains, stabilizes the membrane to local perturbations caused by the DAP insertion. This CL-DAP interaction seems to decrease the ability of the antibiotic to transition from the outer to the inner leaflet, preventing tetramer alignment and pore formation. Both E faecalis and E faecium have adopted strategies to avoid DAP-mediated membrane damage; however, the underlying mechanism seems to be species specific.

In E faecalis, diversion of the antibiotic from critical membrane locations such as the dividing septum is associated with daptomycin resistance (DAP-R). This resistance seems to be associated with redistribution of CL microdomains, which are suggested to act as decoys to sequester the antibiotic away from important septal areas and might also protect the membrane at these locations (the diversion hypothesis).65 Changes associated with this redistribution were described in isolates both in vitro and in vivo and implicated genes involved in the cell envelope stress response (the 3-component regulatory system, LiaFSR) and phospholipid synthesis.66,67 In contrast, E faecium does not exhibit the characteristic redistribution of cardiolipin microdomains seen in E faecalis. Instead, E faecium seems to repel the DAP molecule from the cell surface, changes more akin to the proposed repulsion strategy for DAP resistance described in staphylococci. In this scenario, the positively charged calcium-DAP complex is repelled from the cell surface owing to electrostatic changes (as the cell envelope becomes more positively charged).68 Interestingly, despite the mechanistic differences in DAP resistance between E faecalis and E faecium, similar genes in both species (including liaFSR, see below) are involved.

The lia operon (for Lipid II Interacting Antibiotics) belongs to a family of 3-component signaling systems composed of a sensor histidine kinase (LiaS), its cognate response regulator (LiaR), and a putative transmembrane negative regulator protein (LiaF). The LiaFSR system is conserved in all gram-positive organisms of clinical relevance.69 In the presence of cell envelope stress, the system seems to be activated either by phosphorylation of LiaR (via LiaS) or by mutations in LiaR that mimic phosphorylation.70 In E faecalis, a mutation in liaF (resulting in a deletion of an isoleucine residue at position 177) seemed to activate the system leading to an increase in DAP MIC from 1 to 4 μg/mL. More importantly, this single change in LiaF resulted in loss of DAP bactericidal activity in vitro.71 This increase in MIC and loss of bactericidal activity at a supposedly susceptible MIC questions the current DAP breakpoint for enterococci (4 μg/mL, which is 4 times higher that than for S aureus). DAP-susceptible E faecium isolates with DAP MICs close to the susceptibility breakpoint (3–4 μg/mL) often harbor mutations associated with DAP resistance (mostly in liaFSR),72 and, like E faecalis, these isolates are tolerant (ie, even at 5x, MIC DAP is not bactericidal) to DAP in vitro.73 In a recent report, a patient with recalcitrant E faecium bacteremia treated with DAP experienced failure with this antibiotic, and the initial bloodstream isolate harbored alterations in LiaRS, supporting the notion that changes in LiaFSR are clinically associated with DAP therapeutic failure.2 Of note, mutations associated with DAP resistance have been described in isolates from patients who had never been exposed to DAP, and it is postulated that, in addition to spread from other patients, naturally occurring antimicrobial peptides (like those produced by the innate immune system) may trigger cell envelope adaptive responses similar to those observed with DAP.74,75

The yycFG operon is involved in cell wall homeostasis and is highly conserved among the low G + C gram-positive bacteria. The yycFG operon is known to be active in modification of peptidoglycan synthesis, modulating autolysin expression (including pcsB in streptococci), and mutations in this operon are found in vancomycin-intermediate S aureus and in vancomycin-resistant S aureus strains.76 In addition, the yycFG operon has also been found to have indirect effects on fatty acid metabolism and membrane fluidity. The histidine kinase (YycG) of this 2-component system from S aureus is found to associate with the lipid bilayer via a transmembrane domain and responds to stress via a conserved sensing sequence known as the PAS domain.77,78 Upon activation, the sensor kinase auto phosphorylates before passing the phosphate to its cognate response regulator (YycF) to induce transcription of the regulon. This system was associated with DAP-R in a clinical strain-pair of E faecium that developed resistance to therapy.79 Changes found in the PAS domain were postulated to alter the sensing activity of the YycFG system, although it is not understood precisely how this effect is mediated. Further, in a genomic survey of resistant isolates of E faecium, changes in the yycFG operon were the second most commonly found changes associated with DAP-R, with changes in LiaFSR encountered most often.73

The contribution of alterations in enzymes involved in phospholipid metabolism to the DAP-resistance phenotype has not been fully elucidated, but such changes seem to act synergistically with those related to the cell envelope stress response mediated by LiaFSR (or other regulatory systems, see earlier discussion). Alterations involving enzymes such as cardiolipin synthase (Cls) appear to occur after changes in LiaFSR, potentiating the resistance phenotype.80 Davlieva and colleagues81 recently found that substitutions in one of the phospholipase domains of Cls increased the catalytic activity of the enzyme, albeit marginally. Experimental data have also found that when additional copies of Cls harboring changes found in DAP-R strains were introduced in trans on a plasmid into the laboratory strain of E faecalis OG1RF, they increased the DAP MICs.67 These findings suggest that Cls substitutions are associated with a gain in function of the enzyme; however, how this change is linked to redistribution of CL microdomains is unknown.

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URL: https://www.sciencedirect.com/science/article/pii/S0891552016300125

INTRODUCTION

Daptomycin was originally isolated as a fermentation product of the soil-dwelling bacterium Streptomyces roseosporus. This cyclic lipopeptide antibiotic has recently been licensed in Europe for the treatment of complicated skin and soft tissue infections (cSSTIs) in adults. During the drug development process and beyond, daptomycin has undergone rigorous tests to establish and characterise its antimicrobial activity. The objective of this article is to summarise and discuss the results of these tests, with particular emphasis on the most recent data describing the in vitro activity of daptomycin, its bactericidal activity, protein binding, activity in biofilms, post-antibiotic effect (PAE), synergistic interactions with other antibiotics, and potential for resistance development.

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URL: https://www.sciencedirect.com/science/article/pii/S1198743X14628202

Daptomycin

(t½ 9 h) is a recently released lipopeptide antibiotic, naturally produced by the bacterium Streptomyces roseosporus which was first isolated from a soil sample from Mount Ararat in Turkey.3 It has activity against virtually all Gram-positive bacteria, including penicillin-resistant Streptococcus pneumoniae and MRSA, regardless of vancomycin resistance phenotype. It is unable to cross the Gram-negative outer membrane, rendering these bacteria resistant.

Daptomycin demonstrates concentration-dependent bactericidal activity, including moderately so against most enterococci (for which vancomycin is generally bacteriostatic). Initial binding to the Gram-positive cell membrane is followed by a variety of effects including membrane depolarisation (probably via the drug forming an ion channel across the membrane: this seems to be the main cidal mechanism) and reduced lipoteichoic acid and protein synthesis. A few Clostridium species appear innately resistant, but resistance has proved difficult to induce in vitro and reduction in susceptibility during clinical use has rarely been reported to date. The underlying mechanisms of resistance seem to involve a variety of physiological effects including an altered membrane potential. Staphylococci with increased vancomycin MICs are also less susceptible to daptomycin, and resistance to both agents is acquired progressively in a stepwise fashion.

It is administered by single daily intravenous injection, and is over 90% protein bound. Virtually no metabolism occurs and excretion is predominantly renal, with about 60% of a dose being recoverable unchanged from the urine. The standard dosage is 4 mg/kg per dose, with the frequency of dosing reduced to 48-hourly for patients with creatinine clearances below 30 mL/min. A higher dose of 6 mg/kg/day is being assessed for infective endocarditis. CSF penetration is only about 5%, but sufficient concentrations may be achieved to be useful, for example, for penicillin-resistant pneumococcal meningitis.

Adverse drug reactions have been reported at similar rates to vancomycin. Use of a longer dose interval has avoided the problems of skeletal muscle pain and rises in serum creatinine phosphokinase that were reported when daptomycin was first introduced in the 1980s in a twice-daily regimen – these adverse effects led to its development being interrupted. The effects were were fully reversible and probably related to the need to allow recovery time for drug action on the myocyte cell membrane, but patients receiving daptomycin should nevertheless be monitored for muscle pain or weakness. Weekly serum creatinine kinase assays should be performed during prolonged treatment courses; mild elevations are seen in about 7% of patients and are usually insignificant, but occasionally discontinuation of therapy is needed.

Daptomycin is approved in the UK for treatment of complicated skin and skin structure infections caused by Gram-positive bacteria and right-sided infective endocarditis caused by Staphylococcus aureus (mainly seen in i.v. drug users). Wider applications will doubtless appear and it may prove useful in, for example, endocarditis more generally, osteomyelitis and MRSA infections of orthopaedic hardware. It is usefully employed by outpatient antibiotic therapy clinics because of its single daily dosing and clinical safety. It is not approved for therapy of community-acquired pneumonia because of inferior outcomes which may be related to inhibition by pulmonary surfactant.

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URL: https://www.sciencedirect.com/science/article/pii/B9780702040849000525