Battle against Vancomycin-Resistant Bacteria: Recent Developments

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Battle Against Vancomycin-Resistant Bacteria: Recent Developments in Chemical Strategies Geetika Dhanda, Paramita Sarkar, Sandip Samaddar, and Jayanta Haldar J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01093 • Publication Date (Web): 07 Nov 2018 Downloaded from http://pubs.acs.org on November 9, 2018

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Journal of Medicinal Chemistry

Battle

against

vancomycin-resistant

bacteria:

Recent

developments in chemical strategies Geetika Dhanda, Paramita Sarkar, Sandip Samaddar and Jayanta Haldar* Antimicrobial Research Laboratory, New Chemistry Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bengaluru 560064, Karnataka, India. *Corresponding author: Ph. No.: (+91) 80-2208-2565 Fax: (+91) 80-2208-2627 E-mail: [email protected]

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ABSTRACT Vancomycin, a natural glycopeptide antibiotic, was used as the antibiotic of last resort for the treatment of multidrug-resistant Gram-positive bacterial infections. However, almost thirty years after its use, resistance to vancomycin was first reported in 1986 in France. This became a major health concern and alternative treatment strategies were urgently needed. New classes of molecules, including semi-synthetic antibacterial compounds and newer generations of the previously used antibiotics were developed. Semi-synthetic derivatives of vancomycin with enhanced binding affinity, membrane disruption ability and lipid binding properties have exhibited promising results against both Gram-positive and Gram-negative bacteria. The various successful approaches developed to overcome the acquired resistance in Grampositive bacteria, intrinsic resistance in Gram-negative bacteria and other forms of noninherited resistance to vancomycin will be discussed in this perspective.

1. INTRODUCTION The emergence and spread of antimicrobial resistance (AMR) globally poses a major threat to the management of infectious diseases, resulting in high morbidity and mortality.1 The World Health Organization’s (WHO) Global Antimicrobial Surveillance System (GLASS) revealed high levels of resistance in a number of serious bacterial infections worldwide. Resistance in bacteria such as Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Streptococcus pneumoniae, followed by Salmonella spp are among the most commonly reported.2 To tackle the rapid emergence of antimicrobial resistance, initiatives were taken by various health organizations and governments, which resulted in the approval of various new 2


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antibiotics. Five new classes of antibiotics: lipopeptides (daptomycin), oxazolidinones (linezolid),

pleuromutilin

(retapamulin),

macrocyclic

antibiotics

(fidaxomicin)

and

diarylquinoline (bedaquiline) were introduced into the market. The other new antibiotics, which were modifications of the previously existing antibiotics, had limited usefulness due to the panclass mechanisms of resistance in bacteria. This lack of novel scaffolds from target-based or phenotypic-screening methods had resulted in the withdrawal of pharmaceutical companies from antibiotic research.3 This further aggravated the problem of AMR. Another aspect of bacterial infection that had been given little attention to earlier is the phenomenon of ‘non-inherited’ resistance to antibiotics. In this case, bacteria that are inherently susceptible to antibiotics are often phenotypically resistant to them.4 The ability of bacteria to cause intracellular infections, form biofilms, and go into metabolically inactive state, contributes to this phenotypic ‘noninherited’ resistance. This non-inherited resistance causes an additional burden to AMR and makes treatment more challenging. Among all the classes of approved antibiotics, the glycopeptide antibiotics had the highest longevity in the clinic. The advantage of this class of antibiotics was that they worked by binding to the substrate for peptidoglycan synthesis instead of an enzyme, which is prone to mutations, thereby leading to a decreased propensity of resistance. Vancomycin was reserved as an antibiotic of last resort for the treatment of complicated infections caused by multidrugresistant Gram-positive bacteria which prevented the widespread development of resistance to it. It was not until almost three decades from vancomycin’s introduction in the clinic that resistance to it was observed. The emergence of resistance to this antibiotic raised alarms in the clinic and in the scientific community, calling for immediate measures to curb resistance. According to the list of priority pathogens published by WHO in 2017, vancomycin-resistant Enterococcus

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faecium and vancomycin-intermediate and resistant S. aureus (VISA and VRSA) come under the high priority category since the number of options for the treatment of vancomycin resistant bacteria still remain limited.5 This perspective provides an overview of the current treatment options for vancomycinresistant bacterial infections. The various approaches that have been developed to overcome acquired and non-inherited resistance to vancomycin in bacteria have also been highlighted. Additionally, the strategies to increase the spectrum of activity of vancomycin against Gramnegative bacteria have been discussed.

2. GLYCOPEPTIDE PROGENITORS

Figure 1. Glycopeptide progenitors.

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Glycopeptide antibiotics constitute an important class of cell-wall biosynthesis inhibitors. They are glycosylated heptapeptides produced by a group of soil actinomycetes and are composed of a tricyclic or tetracyclic non-ribosomal peptide core. The glycopeptides are categorized into five different structural subclasses, I-V, based on the amino acid residues for amino acids 1 and 3 in the heptapeptide. Vancomycin, actinoidin A, ristocetin A, teicoplanin and complestatin are some molecules belonging to these subclasses respectively.6 Vancomycin (Figure 1) is the first molecule from this class of antibiotics, which was isolated by Eli Lilly in 1953 and introduced in clinic in 1958.7 Teicoplanin is another clinically relevant molecule which was first reported in 1978 and approved for clinical use in 1988 in Europe.8 Vancomycin contains five aromatic amino acids and two aliphatic amino acids, thus belonging to subclass I of glycopeptide antibiotics. Teicoplanin (Figure 1) contains seven aromatic amino acids and a long fatty acid chain linked to the glucosamine sugar, thus being a lipoglycopeptide. Clinically employed teicoplanin is a mixture consisting of teicoplanin A2-1 to A2-5, with A2-2 being the major component. These drugs, especially vancomycin, are mainstays in the treatment of lifethreatening infections due to methicillin resistant S. aureus (MRSA).8 Vancomycin has been recommended intravenously as a treatment for complicated skin structure infections, endocarditis and bloodstream infections, caused by MRSA. Despite the advantages of vancomycin, there were initially many side effects associated with its intravenous administration, including nephrotoxicity, red-man syndrome and ototoxicity, which have now been resolved due to better purification techniques. It was associated with relatively poor pharmacokinetic (half-life = 4-11 hours) and tissue penetrating properties as compared to other glycopeptide antibiotics. Sometimes, teicoplanin is preferred over vancomycin due to its better potency against various clinical isolates belonging to Enterococcus, Staphylococcus and Streptococcus genera and its

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better tolerability.8, 9 It has better pharmacokinetic properties (half-life = 30 hours) and may be administered intravenously or intramuscularly.

3. MECHANISM OF ACTION The glycopeptide antibiotics inhibit cell-wall biosynthesis by binding to the D-Ala-D-Ala moiety of the peptidoglycan precursors (lipid II and immature peptidoglycan), thus averting transglycosylation and transpeptidation in the later extracytoplasmic stages of cell wall biosynthesis (Figure 2). This complex of glycopeptide antibiotics with the D-Ala-D-Ala terminus is stabilized via five hydrogen bonds, hydrophobic forces and van der Waals forces. Vancomycin is known to form back to back dimers in solution, which results in an improved binding affinity to the substrate. The binding constant of vancomycin with the D-Ala-D-Ala terminus is 4.4 × 105 M-1.10 However, unlike vancomycin, teicoplanin does not form dimers. Additional membrane-anchoring properties are imparted by the lipophilic moiety of teicoplanin which improves association, and thus binding affinity with the substrate. This mechanism of action makes the glycopeptide antibiotics less susceptible to resistance as compared to the enzyme-targeting antibiotics, which are easily rendered ineffective due to genetic alterations. It was due to this unique mechanism of action that vancomycin enjoyed decades of resistance-free use.

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4. RESISTANCE DEVELOPMENT The first instance of vancomycin resistance occurred in Enterococcus faecium in France in 1986.11 In USA, the rise of vancomycin-resistant Enterococci (VRE), a prolific colonizer, gained pace due to the ever-increasing use of vancomycin in hospital settings. In Europe, it happened in communities, probably due to overuse of avoparcin (another glycopeptide antibiotic, used as a growth promoter in livestock).12 Later, in 1996, S. aureus with decreased susceptibility to

Figure 2. Mechanism of action of vancomycin.

vancomycin was first isolated from an infant patient admitted in a hospital in Japan.13 This vancomycin intermediate S. aureus (VISA) strain (Mu50) has been serving as a typical VISA strain with vancomycin minimum inhibitory concentration (MIC) of 8 μg mL-1 since isolation. The first report of high vancomycin resistance in S. aureus which is known as vancomycin resistant S. aureus (VRSA) came in 2002 as a clinical isolate with MIC >32 μg mL-1 accompanied by VRE (vanA positive, vancomycin resistance gene cluster), which was identified

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from a catheter-related infection of a hospitalized patient in USA.14 A strain of S. aureus is considered to be a hetero-VISA (hVISA) strain if it contains subpopulations of cells that grow on the 10-fold more potent than the CBP derivative alone, >1,000-fold more potent than the [Ψ[CH2NH]Tpg4] vancomycin pocket analog, and a striking >10,000-fold more potent than

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vancomycin itself. Recently, in another design, vancapticins were developed by Blaskovich et al. wherein vancomycin was conjugated with moieties that incorporate membrane-disruptive properties. The membrane-interactive moiety consisted of an electrostatic effector peptide sequence (EEPS) and a hydrophobic membrane-insertive element (MIE) (19, Figure 10).84 The details of the various derivatives which

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Figure 9. Lipidation without investigation of membrane-interaction properties (13-16) & incorporation of membrane-disruptive properties (17).

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Figure 10. Incorporation of membrane-disruptive properties.

exhibited activity against vancomycin-resistant strains using this strategy have been covered in Table 1, Figure 9 and Figure 10. 7.1.3 Conjugation of groups to enhance interactions with other components of the bacterial membrane and cell wall Various other components of the bacterial cell membrane can be targeted to inhibit the process of cell-wall biosynthesis. The phosphate group of the C55-lipid (bactoprenol) in the bacterial membrane helps in the transport of the precursor N-acetylglucosamine-N-acetylmuramic acid pentapeptide (GlcNAc-MurNAc pp) from the cytoplasm to the exterior of the cell membrane to

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further the formation of the peptidoglycan chain. Since the target for vancomycin lies in the vicinity of the membrane, conjugating moieties that can bind to the other components of the membrane is an interesting strategy to enhance activity. In an effort to restore the activity of vancomycin in resistant strains, a Zn2+ binding ligand, dipicolyl-1,6-hexadiamine (Dipi) was conjugated to the C-terminus of vancomycin (Dipi-van) by our lab (20 A, Figure 11).85 The idea was that the dipicolyl amine moiety would capture zinc ions (Zn2+), which could be released from apoptotic cells at the site of bacterial infection with high selectivity. The Dipi-van-Zn2+ could then complex with the pyrophosphate of the cell membrane lipids (B, Figure 11). The enhanced inhibition of cell-wall biosynthesis was confirmed by the intracellular accumulation of the precursor, UDP-N-acetylmuramyl-pentadepsipeptide (UDPMurNAc-pp) (C, Figure 11). Dipi-van exhibited a 375-fold and 160-fold increase in activity against VREm (VanA phenotype, E. faecium) and VREs (VanB phenotype, E. faecalis) as compared to vancomycin, respectively (A, Figure 11). Further, this compound reduced the bacterial burden in mice in VRE kidney infection (5 log CFU g reduction). It did not show any increase in MIC when bacteria were subjected to serial passages of the compound indicating no susceptibility to resistance development.

Furthermore, components of the cell wall like the saccharides can also be targeted to enhance the activity of the drug. In another approach to enhance activity by targeting other components of the cell wall, Zhang et al. tethered a benzoxaborole group to the C-terminus of vancomycin, eremomycin and teicoplanin aglycons, as well as to vancomycin; or to the Nterminus of vancomycin and teicoplanin aglycon; or to the amine of vancosamine and Nterminus of vancomycin.86 The rationale behind this was that the benzoxaborole group would

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form additional interactions with the 1,2- and 1,3-diols of saccharides coating the cell surface and therefore enhance the efficacy of the drugs (21, Figure 12).

Figure 11. (A) Structure of Dipi-van. (B) Complex formation of Dipi-van-Zn2+ with geranyl pyrophosphate which was identified through mass spectrometry. (C) Identification of intracellular UDPMurNAc-pp in presence of Dipi-van by monitoring absorbance at λ = 260 nm.

7.2 Glycopeptide hybrids with other antibacterials Antibiotic hybrids provide an interesting approach towards tackling resistance development as they combine the mechanisms of action of different antibiotics. Such hybrids could delay resistance development as genetic alterations in two biological targets are less feasible. In one report, vancomycin–nisin conjugates were developed by linking the C-terminus or N-terminus of vancomycin to the C-terminus of nisin through variable linkers using click chemistry. Nisin is an

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antimicrobial peptide whose N-terminus is known to bind to the pyrophosphate of Lipid II. It was conjectured that through the combination of both moieties in a single molecule, simultaneous lipid II binding of the two parts could lead to increased affinity through bivalency or chelate effect and reinstate the activity of vancomycin to the resistant strains. One of the developed conjugates showed a 40-fold increase in activity against VRE (22, Figure 12).87 Similarly, it was speculated that conjugating a glycopeptide with a β-lactam would result in enhanced antibacterial activity due to the combined mode of action of β-lactams and glycopeptides for cell wall biosynthesis inhibition. With this concept, Theravance Biopharma, Inc. developed vancomycin– cephalosporin hybrids. Out of these, TD-1607 is currently in phase II clinical trials.88,89 Another such hybrid, Cefilavancin (TD-1792) was reported to enter Phase III clinical trials in 2015 but no development has been reported thereafter. These derivatives exhibited enhanced bactericidal activity but were not effective against vancomycin-resistant strains.

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Figure 12. Conjugation of groups to enhance interactions with cell-wall components (21) & vancomycin-nisin hybrid for multiple-target binding (22).

8. TACKLING

INFECTIONS

CAUSED

BY

GRAM-NEGATIVE

BACTERIA Gram-negative bacteria are intrinsically resistant to vancomycin due to the presence of an additional outer membrane composed of lipopolysaccharide. According to the list of priority pathogens published by the WHO in 2017, all the highly critical pathogens are Gram-negative bacteria. Given the dearth of antibiotics to treat infections caused by Gram-negative bacteria, new strategies have to be devised. Various strategies have been employed to extend the spectrum

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of activity of vancomycin to treat such infections (Table 2). Such strategies are welcome as they would provide a new approach to tackle Gram-negative bacteria. This section gives an overview of these strategies. Table 2. Strategies to tackle intrinsic resistance of Gram-negative bacteria to vancomycin.

Strategy

Examples

Activity

A. Incorporation of membrane-active properties

Cationic lipophilic A. baumannii-R4942 derivative of vancomycin (MIC = 3 μM) (17b) E. cloacae: NDM-1 (MIC = 12.5 μM) P. aeruginosa-R590 (MIC = 6.1 μM) B. Adjuvant therapy  Employment of silver 90% survival of mice (Ag+) as an adjuvant with in an acute mouse peritonitis E. coli vancomycin infection model P. aeruginosa (MIC  Polymyxin-tobramycin vancomycin hybrids as adjuvant with of reduced to 0.5 μg mLvancomycin 1)  Colistin as an adjuvant A. baumannii (MIC of teicoplanin ≤ 2 μg with teicoplanin mL-1)  Colistin as an adjuvant MIC of telavancin < 1 μg mL-1 against with telavancin clinical isolates  Nitrofurantoin as an E. coli (MIC of adjuvant with vancomycin vancomycin = 12.5 μg mL-1)  Trimethoprim as an E. coli (MIC of adjuvant with vancomycin vancomycin = 6.25 μg mL-1) 2+ C. Vancomycin Zn binding Dipi-van as a MIC of meropenem derivative as metalloβ-lactamase reduced to 3.15 μg metallo-βinhibitor used in mL-1 against NDM-1 lactamase combination with producing Graminhibitor meropenem (A, Figure 14) negative pathogens D. Other approaches

References 90

91

92

94 95 96 96 97

 Loading vancomycin into E. coli and A. 98 fusogenic liposomes baumannii (MIC = 6 liposomes composed of μg mL-1) phospholipid–cholesterol

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hemisuccinate mixture  Conjugation vancomycin with nanoparticles

of E. coli (8 μg mL-1) gold

99

8.1 Membrane-active semi-synthetic derivatives of vancomycin The cationic lipophilic analogue of vancomycin (17b, Figure 9) displayed remarkable activity against Gram-negative pathogens, including carbapenem-resistant clinical isolates with MICs ranging from 1.2-6.1 μM.90 It could permeabilize and depolarize the outer and inner cell membranes of E. coli, proving that the compounds compromised the cell membrane integrity. Compound 17b did not induce resistance in A. baumannii, although the currently used antibiotic, colistin showed an increase in MIC from the fifth passage of antibiotic exposure itself. It exhibited good in-vivo antibacterial activity against A. baumannii in mouse thigh infection model (3 Log CFU g-1 reduction in bacterial load). Thus, conjugation of cationic lipophilic moieties with a permanent positive charge was proved to be effective even in clinically relevant Gramnegative pathogens due to an additional membrane-disruptive mechanism of action.

8.2 Adjuvant therapy Another approach to tackle resistance against vancomycin is through ‘adjuvant therapy’ or ‘combination therapy’. An adjuvant is an agent that modifies the effect of other agents at comparatively lower concentrations. Small molecules and nanoparticles have been used as adjuvants to potentiate the activity of vancomycin or other glycopeptides against the resistant strains of bacteria. Silver has been known to exhibit antibacterial activity through permeabilization of the bacterial membrane. Induced membrane permeability could sensitize

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various intrinsically resistant strains of bacteria including Gram-negative pathogens to vancomycin. Collins' group reported in 2013 that on using silver as an adjuvant, Gram-negative bacteria were sensitized to vancomycin in-vitro.91 They demonstrated that Ag+ potentiated vancomycin against E. coli in a mouse peritonitis model. More recently, polymyxin-tobramycin antimicrobial hybrids, which show potent antibacterial activity (MIC of 2−16 μg mL-1 or 0.7−5.7 μM) against multidrug resistant/extensively drug resistant (MDR/XDR) P. aeruginosa strains have also been demonstrated to act as an adjuvant with various antibiotics against P. aeruginosa.92 The individual components of the hybrid were conjugated through Coppercatalyzed alkyne azide click reaction (CuAAC), a 1,3-dipolar cycloaddition followed by removal of the protecting groups. Vancomycin, which is ineffective in Gram-negative bacteria due to the impermeability of the outer membrane, was potentiated in P. aeruginosa strains due to the synergistic membrane-disruptive activity of the antimicrobial hybrid. The synergistic mechanism was proposed due to Fractional Inhibitory Concentration Index (FICI) value of

Battle against Vancomycin-Resistant Bacteria: Recent Developments

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