Glycopeptide antibiotics and development of inhibitors to overcome vancomycin resistance

Contents

• 1 Introduction
  • 1.1 Vancomycin and other key glycopeptide antibiotics

• 2 Molecular basis for vancomycin resistance in Enterococcus faecium

• 3 Development of inhibitors of vancomycin-resistant enzymes as new antibiotics
  • 3.1 Inhibition studies of VanR and VanS
  • 3.2 VanA as a target for new inhibitors
  • 3.3 Inhibitors of VanX and VanH

• 4 Prospects

• References

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Abstract

Covering: Up to July 2001.

This review outlines the history of vancomycin and other key glycopeptide antibiotics and the molecular basis of vancomycin resistance, and focuses on the development of synthetic inhibitors of vancomycin-resistant enzymes VanR, S, A, H and X to overcome resistance. The literature up to July 2001 is reviewed and 119 references are cited.

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Yong Gao

Dr Yong Gao was born in 1969 in Luan, China. He obtained his B.Sc. in 1990 from the Chemistry Department, Wuhan University, China. In 1993, he moved to Canada to pursue his graduate studies in the Department of Chemistry at the University of Alberta, where he received his Ph.D. in 1998 under the supervision of Prof John C. Vederas. He then joined Prof Robert R. Rando's group in the Department of Biological Chemistry and Molecular Pharmacology at Harvard Medical School as a postdoctoral fellow. In 2000, he was appointed as a tenure-track assistant professor in the Department of Chemistry and Biochemistry, Southern Illinois University Carbondale. Dr. Gao's research interests are in the areas of developing new antibacterial agents targeting bacterial peptidoglycan structures and synthesis of nano- and bio-materials for biological applications.

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1 Introduction

Vancomycin and teicoplanin are members of the glycopeptide antibiotic family which are in clinical use today for bacterial infections caused by Gram-positive pathogens (Fig. 1).¹ However, after four decades of vancomycin clinical use, resistant pathogens to vancomycin have appeared. In 1988, the emergence of vancomycin-resistant enterococci (VRE) was recognized for the first time.^2,3 Further, since 1997 vancomycin-resistant Staphylococcus aureus strains have been found in different areas.^4,5 Given the fact that vancomycin is often the last therapeutic agent in the treatment of methicillin-resistant S. aureus infections,⁶ bacterial resistance to vancomycin poses a serious health threat.

Fig. 1 Glycopeptide antibiotics.

In the quest to find antibiotics to combat vancomycin resistance, three strategies have been developed. The first strategy is to design new antibiotics that interact with novel targets in the bacterial life cycle that conventional antibacterials are not aimed at. The oxazolidinone linezolid (Zyvox) approved last year by the U.S. FDA is an example of a new class of antibiotics.⁷ The second strategy is to examine analogs of vancomycin glycopeptides which are produced through chemical, biosynthetic and new isolation efforts. Recent achievements in the total chemical synthesis of vancomycin,^8,12 the vancomycin aglycon,^9–16 and modifications in the vancomycin carbohydrate unit^17,18 have brought new synthetic routes to more effective versions of glycopeptide antibiotics. N-(Chlorobiphenyl)chloroeremomycin, LY333328¹⁹ from Eli Lilly company in Indianapolis and a similar compound²⁰ from Lepetit in Milan, Italy, represent the successful application of this approach. Readers are directed to a recent review paper by Nicolaou²¹ for developments in this area. The biosynthesis of vancomycin glycopeptides is a very active and important research area, bringing promise of producing novel glycopeptide analogs by combinatorial biosynthesis. Williams²² has reviewed developments in this area in 1998 and several recent papers published afterwards are listed here^23–29 for those readers who are interested in vancomycin biosynthesis. The third approach is to design inhibitors that target vancomycin-resistant enzymes utilized by VRE, modifying the cell wall structure resulting in vancomycin resistance. Deactivation of these enzymes will intervene and reverse vancomycin resistance. The mechanism of vancomycin resistance has been extensively reviewed^{21,22,30–34} and the present discussion focuses on new inhibitors of vancomycin-resistant enzymes.

1.1 Vancomycin and other key glycopeptide antibiotics

Vancomycin was first isolated from a soil sample collected by Eli Lilly company in the mid-1950s.³⁵ It is produced by the microorganism Streptomyces orientalis and was first used clinically in 1959. Since then, vancomycin has proved to be active against most Gram-positive bacteria and is used in the treatments of infections due to staphylococci (including methicillin-resistant Staphylococcus aureus), streptococci, and enterococci.

The structural elucidation of vancomycin represents an impressive achievement for decades. Most notable were: in 1965, Marshall³⁶reported structural studies of vancomycin through degradative analysis; in 1977, Williams³⁷ obtained a partial structure of vancomycin by using NMR spectroscopy; in 1978, Sheldrick³⁸used X-ray crystallography to analyze the degradative product of vancomycin; in 1982, Harris³⁹ proposed the structure of vancomycin; the crystal structures of complete vancomycin⁴⁰ and parvodicin aglycon⁴¹ were solved by Sheldrick in 1996. The mode of action of vancomycin was proposed by Williams^37,42 and confirmed by X-ray crystallographic studies carried out by Loll and Axelsen in 1997⁴³ and 1998.⁴⁴ Recently, the total synthesis of vancomycin aglycon has been completed and reported from the groups of Evans,^13,14 Nicolaou^9–11 and Boger^15,16 and the total synthesis of vancomycin from the aglycon has been achieved by Nicolaou^8–12 and Kahne.¹⁷

Vancomycin contains a peptide backbone constituted of seven amino acids designated as 1 to 7 from the N- to the C- terminus (Fig. 1).⁴⁵ The five aromatic rings are lettered A through E. The stereochemistry at the α-C centers of the amino acids 1 to 7 are R,R,S,R,R,S,S. Chlorine atoms are attached to aromatic rings C and E, and a disaccharide is linked to the aromatic ring D. The vancomycin aglycon is a trimmed version of vancomycin without the carbohydrate units (Fig. 1). Teicoplanin and ristocetin A are naturally occurring glycopeptide antibiotics, both having a heptapeptide backbone (Fig. 1). Recently, natural and semisynthetic analogs of vancomycin are adding to the glycopeptide family through new isolations, and chemical and biological syntheses. Among them, vancomycin and teicoplanin are the only two currently used clinically.

2 Molecular basis for vancomycin resistance in Enterococcus faecium

The netlike peptidoglycan layer of the bacterial cell wall provides much of its strength and rigidity and is vital to the organism's survival.^33,46 Vancomycin acts by binding to the bacterial cell wall precursor that terminates in the sequence D-Ala-D-Ala²² (Fig. 2), which prevents subsequent transglycosylation and transpeptidation. Failure to attach the disaccharyl pentapeptide and to execute a crosslink lowers the interstrand covalent connectivity of the peptidoglycan layer, decreasing its mechanical strength and leading to cell lysis due to high internal osmotic pressure.^{31,32,47–51}

Fig. 2 Binding of vancomycin and the D-Ala-D-Ala terminus of a peptidoglycan precursor. Vancomycin binds with high affinity to the terminus of the lipid-PP-disaccharide-pentapeptide intermediate through a network of five hydrogen bonds.

VRE are clinically divided into five categories: types A, B, C, D and E.^31,52–61 The molecular mechanism of VanA-type resistance has been well characterized, largely due to research work of Courvalin^31,50,51,56 and Walsh.^30,32,50,51 It is now known that VRE type A expresses its resistance through five plasmid-born genes vanS, vanR, vanH, vanA and vanX.^30,32 Gene products of vanR and vanS form a two-component signal transduction system that activates the expression of genes vanH, A and X (Fig. 3). VanS is a trans-membrane sensor kinase that undergoes phosphorylation at a His residue (His-164) upon activation by external stimuli. This product in turn causes phosphorylation of VanR, which binds to the promoter region of vanH and induces expression of genes vanH, A and X. VanH is a pyruvate reductase that generates D-lactate (Fig. 4). VanX is a dipeptidase that hydrolyzes D-Ala-D-Ala to produce D-alanine. VanA utilizes D-alanine and D-lactate to synthesize D-Ala-D-Lac which is transported and incorporated into the modified peptidoglycan structure outside the cytoplasm. This results in a modified peptidoglycan of VRE terminating in D-Ala-D-Lac in place of D-Ala-D-Ala which is synthesized by the gene ddls in E. coli. The mutation of the terminal amino acid of the peptidoglycan from D-alanine to D-lactate in VRE results in a 1000-fold decrease in vancomycin binding affinity and in unimpeded peptide cross-linking.⁵⁰ The outcome is a mechanically strong cell wall whose biosynthesis is not inhibited by vancomycin glycopeptides.

Fig. 3 VanRS two-signal transduction system. VanS is a trans-membrane sensor kinase that undergoes phosphorylation at a His residue upon activation by external stimuli. This product in turn causes phosphorylation of VanR, which promotes transcriptional activation and expression of the genes vanH, A and X.

Fig. 4 Synthesis of D-Ala-D-Lac. Proteins VanH and VanX provide the starting materials D-Lac and D-Ala that are utilized by VanA to synthesis D-Ala-D-Lac as part of the modified peptidoglycan structure.

VanB-type VRE acts in a fashion similar to VanA type by replacing the terminal dipeptide D-Ala-D-Ala from the pentapeptide precursor with D-Ala-D-Lac.³¹ In the case of VanC, D-Ala-D-Ser is substituted for D-Ala-D-Ala.^52–54 The VanA-type strains have high-level resistance to vancomycin and teicoplanin, and VanB-type strains are characterized as having various levels of vancomycin resistance and being susceptible to teicoplanin. Type C resistance is significantly different from types A and B. It presents only modest resistance to vancomycin and teicoplanin and appears to be chromosomal rather than plasmid-born.^{52,54,58–61} VanD and VanE clinical phenotypes have recently been reported.^55,56

3 Development of inhibitors of vancomycin-resistant enzymes as new antibiotics

VanA-type VRE is the most common vancomycin resistant species found in clinical isolates. The molecular logic of each of the five vancomycin-resistant proteins revealed opportunities to intervene and convert vancomycin resistance back to sensitivity by inhibiting VanR, S, H, A and X.³⁰

3.1 Inhibition studies of VanR and VanS

VanR and VanS comprise a two-component signal transduction system which regulates vancomycin resistance in VRE (Fig. 3).^62,63 VanS, the sensor kinase which detects the external stimuli, undergoes a net increase in the level of phosphorylation of one of its residues (His-164) by ATP.⁶⁴ VanS then transfers the phosphoryl group from His-164 of VanS to Asp-55 on VanR, the response regulator for vancomycin resistance, therefore causing P-vanH to activate the expression of the vanHAX gene cluster.^65–67 Although VanR is a preferred phosphoryl acceptor, VanS can have a cross-talk with the heterogeneous (E. coli) response regulator PhoB.^68–70 The detailed mechanism of activation of VanS is not clear, but experiments suggested that vancomycin resistance probably is induced by altering the synthesis/degradation balance of the cell wall,^71–74 which stimulates the phosphorylation of VanS. The two-component regulatory systems of bacteria are logical targets for chemotherapeutic intervention⁷⁵ because homologous proteins have been identified only in yeast and fungi^76–79 and not in higher eukaryotes.⁷⁵ The VanRS system presents opportunities to block the relay of the resistance signals and stop the expression of vancomycin-resistant enzymes VanA, H and X.

Halophenyl isothiazolone LY-266,400 (1) (Fig. 5), a compound shown to reduce expression of the AlgR1-AlgR2 two-component signal transduction pathway in Pseudomonas aeruginosa,⁸⁰ was recently found to be an inhibitor of the VanRS system by Weisblum.⁸¹ This compound attenuated the ability of VanR to accept a phosphoryl group from VanS∼P with an ED₅₀ (50% effective dose) of 0.35 mM and the accumulation of VanS∼P was observed. Analog LY-266,408 (2) had an effect similar to inhibitor 1.

Fig. 5 Isothiazolone inhibitors of the VanRS system.

Macielag et al.⁸² reported salicylanilides 3 and 4 (Fig. 6) as inhibitors of the VanRS pathway. Salicylanilide 3 inhibited both the growth of vancomycin-resistant E. faecium OC3312 and the VanS/VanR dependent expression of VanH-luc utilized by a whole-cell reporter-gene assay in a concentration-dependent fashion. This test system, similar to the ones developed by the groups of Walsh,^66,67 Wanner^66,67 and Weisblum,⁷³ consists of an Enterococcus faecalis cell line which expresses the VanRS mediating vancomycin resistance and VanH-luc, a fusion protein comprised of VanH and firefly luciferase (luc). The effects of compound 3 on luciferase expression could be detected at concentrations as low as 0.5 μM. Compound 4 also inhibited VanR/VanS in the whole cell at concentrations subinhibitory for growth. Interestingly, although they inhibited the bacterial cell growth with minimal inhibitory concentrations (MICs) ranging from 0.5 to 2 μg mL⁻¹, analogs 5–8 actually activated the VanRS to induce overexpression of luciferase specific activity. The strong antibacterial activity of salicylanilides 5–8 is probably due to their effects on multiple targets in the whole cell. Although unlikely to be effective chemotherapeutic agents themselves due to their effect on mitochondria respiration, salicylanilides 3 and 4 may provide a structural template for the design of the next generation of selective inhibitors of the two-component signal transduction systems.

Fig. 6 Salicylanilides.

A systematic search for inhibitors of signal transduction reported by Barrett et al.⁸³ led to the new antibacterial agents RWJ-49815 (9) and its analogs 10 and 11 (Fig. 7). These compounds inhibited the growth of Gram-positive pathogenic bacteria, including vancomycin-resistant E. faecium OC3312 with MICs of 1–8 μg mL⁻¹. Compound 12 was a less effective agent against VRE with an MIC over 32 μg mL⁻¹. Tyramines 9–11 interacted with VRE, presumably as inhibitors of the VanRS system since they all inhibited the autophosphorylation of kinase A of the KinA–Spo0F two-component transduction system. The IC₅₀ values of 9, 10 and 11 against kinase A are 1.6 μM, 20 μM and 14 μM, respectively.

Fig. 7 RWJ-49815 and analogs against vancomycin-resistant E. faecium OC3312.

Recent work by Weisblum and coworkers⁸⁴ have found that peptide agents can also inhibit the VanRS two-component signal transduction process. The dodecamer peptide E12 (SLCHDSVIGWEC) was selected on the basis of its ability to bind to VanR protein from a phage library displaying combinatorial peptides. E12 inhibited the formation of complexes of VanR-PvanH and VanR∼P- PvanH with IC₅₀ values of 3 μM and >1 mM, respectively. The binding of E12 to VanR was localized to the N-terminal, regulatory domain of VanR consisting of Asp-55. With a single gap, E12 matches the 18-amino acid sequence, residues Tyr-161 to Ser-178, which is part of the catalytic center dimerization domain of VanS, a sequence that VanR also normally interacts with. A modified peptide E12.1 (SLAHDSIIGYLS), designed based on E12 and more closely resembling the sequence of VanS, inhibited the binding of VanR∼P to P_vanH with an IC₅₀ of 100 μM. Alanine substitution analysis supports the hypothesis that E12 binds to VanR by mimicking the key interaction site of VanS.

3.2 VanA as a target for new inhibitors

VanA protein in VanA-type VRE is a D-Ala-D-Lac ligase which takes D-alanine and D-lactate subsequently as substrates (Fig. 4).^51,85 Similar depsipeptide ligases have been found in vancomycin producers,^86,87 the lactic acid bacteria and soil organisms with natural resistance to vancomycin producers, including LmDdl2 ligase from Leuconostoc msenteroides.⁸⁸ In Gram-negative E. coli, Ddls are used for the formation of D-Ala-D-Ala for the unaltered peptidoglycan structure. VanA and DdlB share about 28% sequence identity and the same phosphorylated D-alanine intermediate during the ligation process,⁸⁹ but they show different specificities for the second ligands: VanA adopts D-lactate; DdlB takes D-alanine (Fig. 8).^90,91 Considerable efforts have been made by Walsh and coworkers on understanding the substrate specificities of these two ligases^{51,60,90–98} and recent crystallographic studies by the groups of Knox^99–101 and Roper^102,103 have brought more insights on their structural–functional relationship. A recent review by Healy et al.¹⁰⁴ has summarized developments in this area.

Fig. 8 Formation of D-Ala-D-Ala and D-Ala-D-Lac by DdlB and VanA. A D-alanyl phosphate is the common intermediate for the two ligases followed by selective recognition for the second substrate.

The phosphinate dipeptide analogs 13–15 and phosphonate analogs 16 and 17 (Fig. 9) were prepared and tested against DdlA, DdlB and VanA by Bartlett and coworkers.¹⁰⁵ The K_i values in columns labeled “A” in Table 1 correspond to an inhibitor competition with both substrates binding to a free enzyme while the K_i′ figures in columns labeled “B” represent competition with the second substrate binding to the complex of ligase–the first substrate D-alanine. Although many of the compounds 13–17 inhibited wild-type DdlA and DdlB strongly, all of the tested molecules showed reduced affinity to VanA. Based on the experimental results, Bartlett has proposed mechanisms for the ligation reactions carried out by Ddls and VanA, in which proton transfer from the attacking nucleophile to the departing phosphate occurs directly without intervention of the enzymes. X-Ray crystal structures of the phosphorylated form of 13 incorporated in DdlB, Y216F mutant of DdlB, LmDdl2 ligase and VanA have been reported by Knox, Walsh and Roper.^99–103

Fig. 9 Phosphinates and phosphonates.

Table 1 Inhibition constants of phosphinates and phosphonates 13–17 with DdlA, DdlB and VanA

Compound number^a	DdlA		DdlB		VanA
	A^b /nM	B^c /μM	A/nM	B/μM	A/nM	B/μM
a Structures of phosphinates and phosphonates can be found in Fig. 9. b A: Reversible inhibition Ki values of ligases by phosphinate and phosphonate analogs binding to free enzyme. c B: Reversible inhibition Ki′ values of ligases by phosphinate and phosphonate analogs binding to ligase–D-Ala1 complex.
13	55	27	3	4	4100	60
14	18	9	3	4	1600	23
15	6	3	2	3	750	11
16	1600	800	12	18	8800	130
17	2000	100	13	20	6800	100

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3.3 Inhibitors of VanX and VanH

VanX is a novel Zn-dependent D,D-dipeptidase^106,107 which is essential for vancomycin resistance in E. faecium to provide D-alanine for reprogramming the bacterial cell wall (Fig. 4).^108,109 X-Ray crystallography has shown that a zinc ion occupies a central position inside the cavity and is coordinated with three residues and a water molecule.¹¹⁰ In the proposed reaction mechanism, the water molecule displaced by the entry of the substrate D-Ala-D-Ala is activated by Glu-181 and attacks the zinc-polarized carbonyl of D-Ala-D-Ala hydrolyzing the amide bond.^110–112 Competitive ligands binding to zinc ion replace the water molecule and/or the binding residues in the active site, resulting in VanX inhibition.

Indeed, Walsh and coworkers have identified a group of dithiol compounds (Table 2), most of which showed potent, time-dependent inhibition of VanX, presumably by acting as binding ligands to the zinc ion (Fig. 10).¹¹³ Among the dithiols tested, 2,3-dimercaptopropane-1-sulfonic acid and 2,3-dimercaptopropan-1-ol are the most potent, with K_i* values 10⁴-fold higher than substrate (K_m of 1.4 mM). Walsh's group has also assayed several monothiol compounds, but their inhibition activities are less striking.

Table 2 Inhibition constants of dithiols against VanX

	K i* /μM^a
a The final inhibition constant Ki* was estimated by Dixon plot of steady-state kinetics.
Ethane-1,2-dithiol	1.8
2,3-Dimercaptopropan-1-ol	0.32
2,3-Dimercaptopropane-1-sulfonic acid	0.19
Propane-1,3-dithiol	17
Dithiothreitol	7.3

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Fig. 10 Inhibition of VanX by dithiols. Dithiols act as bidentate ligands to zinc mimicking a five-membered transition-state analog of the natural substrate D-Ala-D-Ala.

Another class of inhibitors that has been studied by Walsh's group on their ability to deactivate VanX protein are the phosphinate dipeptides.¹¹⁴ Phosphinate 13 (Fig. 9), known as a potent inhibitor of Ddls,¹⁰⁵ showed a time-dependent onset of inhibition of VanX with a final K_i* of 0.47 μM followed by a time-dependent return to uninhibited steady-state rates upon dilution of the enzyme–inhibitor mixture. Phosphinates 18 and 19 (Fig. 11) are racemic mixtures and their D,D-analogs, inhibiting VanX with estimated K_i* values of 90 nM and 0.44 μM, respectively. Phosphinate 13 has also been studied by Crowder's group using a new continuous assay which employs L-alanine-p-nitroanilide as a substrate instead of natural substrate D-Ala-D-Ala.¹¹⁵

Fig. 11 Phosphinate inhibitors of VanX.

Recently, a fluorine dipeptide 20 (Fig. 12) has been identified as a potent inhibitor of VanX by Aráoz et al.¹¹⁶ The inhibition studies suggested that VanX-mediated peptide cleavage generates a highly reactive 4-thioquinone fluoromethide 22 which is able to covalently react with enzyme nucleophilic residues, resulting in irreversible inhibition. Deactivation was associated with the elimination of fluoride ion as deduced from ¹⁹F NMR spectroscopy analysis and with the production of fluorinated thiophenol dimer 21. Inhibition of VanX by 20 was time-dependent with K_irr and k_intact values of 30 ± 1 μM and 7.3 ± 0.3 min⁻¹, respectively. The deactivation of 20 was also active site-directed, as shown from substrate protection experiments.

Fig. 12 The inactivation mechanism of VanX by dipeptide 20 proposed by Aráoz et al. 4-Thioquinone fluoromethide 22 is an intermediate which reacts with enzyme nucleophilic residues forming a covalent bond. The mechanism was supported by the isolation of a thiophenol dimer 21.

VanH is an α-keto dehydrogenase that stereospecifically reduces pyruvate to D-lactate (Fig. 4),³⁰ which is homologous to VanHst from the glycopeptide antibiotic producing organism¹¹⁷ and 2-hydroxyacid dehydrogenase.¹¹⁸ Oximate was reported to be a competitive inhibitor of pyruvate for VanHst with K_i of 28 ± 3 μM.¹¹⁷

4 Prospects

Vancomycin resistance is a serious health threat and there is an urgent need to produce new antibiotic drugs to fight resistant strains. Unfortunately to date no potent in vivo inhibitors of vancomycin-resistant enzymes have been found. The inhibitors of VanH, A and X discovered so far are amino acid and dipeptide analogs and poor cellular uptake of these molecules may contribute to their low in vivo activity.³⁰ With recent knowledge gained on the resistant enzymes, especially the X-ray crystal structures of VanA and VanX,^99–102,110 new efforts on designing novel, non-polar inhibitors of the resistant enzymes may lead to antibiotics that will combat VRE. VanRS are good targets for developing new antibacterial agents as two-component signal transduction systems have yet to be found in animal cells. VanR is probably a more attractive target due to a recent report that deactivation of its homologous enzyme VncR in S. pneumoniae by site-specific recombination-mediated gene disruption is lethal.¹¹⁹

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