Oritavancin Retains a High Affinity for a Vancomycin-Resistant Cell

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Oritavancin Retains High Affinity to a Vancomycin-Resistant Cell-Wall Precursor via its Bivalent Motifs of Interaction Sierra Bowden, Christine Joseph, Shengzhuang Tang, Jayme Cannon, Emily Francis, Michelle Zhou, James R. Baker, and Seok Ki Choi Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00187 • Publication Date (Web): 13 Apr 2018 Downloaded from http://pubs.acs.org on April 13, 2018

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Biochemistry

Oritavancin Retains High Affinity to a VancomycinResistant Cell-Wall Precursor via its Bivalent Motifs of Interaction Sierra Bowden,‡ Christine Joseph,‡ Shengzhuang Tang,†, ‡ Jayme Cannon,†, ‡ Emily Francis,‡ Michelle Zhou,‡ James R. Baker, Jr.,†, ‡ and Seok Ki Choi*, †,‡ †

Department of Internal Medicine, and ‡Michigan Nanotechnology Institute for Medicine and Biological Sciences, University of Michigan Medical School, Ann Arbor, Michigan 48109, United States

*To whom correspondence should be addressed, Phone: (734) 647-0052; Fax: (734) 936-2990; Email : [email protected]

ACS Paragon Plus Environment

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ABSTRACT

Despite its potent antibacterial activities against drug-resistant Gram(+) pathogens, oritavancin remains partially understood in its primary mode of hydrogen bond interaction with a cell-wall peptide regarding the role of its lipophilic 4′-chlorobiphenyl moiety. Here we report a surface plasmon resonance (SPR) study performed in two cell-wall model surfaces, each prepared by immobilization with a vancomycin-susceptible Lys-D-Ala-D-Ala or vancomycinresistant Lys-D-Ala-D-Lac peptide. Analysis of binding kinetics performed in the peptide surface showed that oritavancin bound ~100 to 1000–fold more tightly than vancomycin in each model surface. Ligand competition experiments conducted by SPR and fluorescence spectroscopy provided evidence that such affinity enhancement is attributable to its 4′-chlorobiphenyl moiety, possibly through a hydrophobic interaction which led to a gain in free energy with a contribution from enthalpy as suggested by a variable-temperature SPR experiment. Based on these findings, we propose a model for the bivalent motifs of interaction of oritavancin with cell-wall peptides, by which the drug molecule can retain a strong interaction even with the vancomycin-resistant peptide. In summary, this study advances our knowledge of oritavancin and it offers a new insight on the significance of bivalent motifs in the design of glycopeptide antibiotics.

Key words: Oritavancin, Vancomycin, Drug Resistance, Cell-Wall Peptide, Hydrogen Bond, Hydrophobic Interaction, Surface Plasmon Resonance Spectroscopy


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Biochemistry

Introduction The vancomycin (VAN) class of glycopeptide antibiotics constitutes drugs of last resort in the treatment of serious bacterial infections caused by multidrug-resistant Gram(+) pathogens.1, 2 The key mechanism of action by which these drug molecules kill bacterial cells involves the blocking of cell-wall biosynthesis and maturation at the stage of transpeptidation and transglycosylation.1,

3-5

It occurs through drug binding to D-Ala-D-Ala, a cell-wall peptide

precursor terminated in the peptidoglycan (PG) strand, through the formation of intermolecular hydrogen bonds (HB) (Figure 1). Of these drugs, oritavancin (ORI)6 is of particular importance because it displays more potent antibacterial activities by two to three orders of magnitude than VAN against Gram(+) pathogens including drug-resistant S. aureus and E. faecium.6, 7 Due to its potent activities, ORI serves as one of the important leads in the drug optimization and design of new glycopeptide antibiotics.4, 8-10 ORI has been a subject of active investigations for its modes of action.11-15 As a semisynthetic variant of VAN, ORI retains an identical aglycon structure that interacts with the cell-wall peptide backbone, which is responsible for its primary HB interaction. However, it has a structural feature absent in VAN with a lipophilic 4'-chlorobiphenyl methyl group which is attached to its (R)-epi-vancosamine sugar unit. In the early stage of its development, ORI was reported to display similar binding affinity to cell-wall precursor peptides compared to VAN.6 More specifically, the solution study conducted by affinity capillary electrophoresis suggests that both VAN and ORI show the same µM affinity to Ac-Lys-D-Ala-D-Ala (KD = 2–4 × 10–6 M) while both show only mM affinity to Ac-Lys-D-Ala-D-Lac (KD = 2–4 × 10–3 M), which forms the basis for VAN resistance in certain drug-resistant pathogens.


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Figure 1. Structure of vancomycin (VAN) and oritavancin (ORI), and their primary mode of action. In VAN-susceptible Gram(+) bacteria, the drug action occurs through a concerted array of five hydrogen bond (HB) pairs formed between the drug molecule and a cell-wall precursor peptide.3 However, in VAN-resistant bacterial cells, it is disrupted due to a reprograming in the precursor peptide from D-alanine to D-lactate, which results in a repulsive dipole-dipole interaction.16 Later, a few mechanism of action studies have been performed for ORI in order to better understand its superior activities against VAN-resistant bacterial pathogens.6,

7, 12, 17

Each of

these was closely associated with a functional role played by its 4′-chlorobiphenyl moiety (Figure 2),11, 14 and it occurs at the cell-wall site (secondary site) located deeper than the peptide precursor (primary site). These sites include a penta-glycine crosslink bridge in close proximity to the peptide precursor in nascent and immature PG strands (Kim, et al., and Schaefer, et al.),12,


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Biochemistry

18-20

a membrane-bound lipid II peptide (Schneider, et al.),7 and the cell membrane as the site for

drug dimerization and anchoring which is known to promote membrane permeability and depolarization (Allen, et al.,6 Ndieyira, et al.,21 and Mingeot-Leclercqa, et al.17). However, despite the significance of these modes of action,7,

12, 17, 19

it remains unclear how drug

accumulation and penetration can occur to such inner sites, in particular, in the VAN-resistant cell wall. For example, these actions would not occur effectively if the drug molecule only weakly binds in the upper PG layer due to its low affinity (KD = 4 × 10–3 M) as reported earlier.6 In this present article, we are interested in clarifying such inconsistency and advancing our knowledge of the modes of action for ORI occurring on the growing cell wall.

Figure 2. A schematic illustration for the multiple modes of action displayed by ORI in the VAN-susceptible (A) and VAN-resistant (B) cell walls. Those marked within the dotted circles constitute the focus of this study. Our present study aims to evaluate the affinity of ORI and validate its new mode of action in the cell-wall model surface. It describes surface plasmon resonance (SPR) spectroscopy22, 23 used


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for evaluating the binding kinetics of VAN and ORI in two cell-well models, each immobilized with either a VAN-susceptible or VAN-resistant peptide. This SPR approach offers a unique ability to study the primary HB interaction alone in the surface using an essentially minimal peptide precursor required for drug recognition.22, 24 It does not contain a secondary binding site such as the penta-glycine crosslink residue,12 lipid II,7 or the hydrophobic lipid membrane,6 and thus it allows to attribute the binding kinetics entirely to the HB interaction between ORI and the peptide. This present study is focused on three aspects. First, the binding kinetics of each drug molecule performed on the surface is described to compare their values of affinity constant measured under an identical condition, and to show the ability to retain an affinity by ORI in the VAN-resistant surface. Second, this study presents evidence important for defining the structural basis of their distinct affinity with a plausible model proposed for ORI. In this aspect, the 4′chlorobiphenyl moiety in ORI is presented as a hydrophobic motif which could potentially contribute to its greater affinity. This aspect is discussed with the results of competitive ligand displacement experiments performed by co-incubation with beta-cyclodextrin (β-CD).25 Third, a thermodynamic aspect in the drug-peptide interaction is also investigated by variabletemperature SPR with a focus on the extent of an enthalpic gain contributed by 4′-chlorobiphenyl underlying the bivalent motifs of interaction by ORI. In summary, the findings of this current study suggest new knowledge and insights on the mode of action by ORI. We believe that they provide supportive evidence of drug binding in the PG layer for penetration and access to other inner sites of action reported for ORI.11, 14 Results and Discussion


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Biochemistry

Peptidoglycan Cell-wall Models. This study employed SPR spectroscopy and investigated the kinetics of drug-peptide interactions occurring on the model surface of the PG layer.22, 24, 26 As one of a few biophysical methods that allow the measurement of the kinetics for receptor– ligand interactions on the surface,27 SPR spectroscopy has played a critical role in defining the affinity and mechanisms of drug action for antibiotic molecules, including VAN.22,

26

In this

study, two bacterial cell-wall models were designed in a CM5 sensor chip for SPR. Each of these was prepared by the amide coupling of a cell-wall precursor peptide, Nα-Ac-Lys-D-Ala-D-Ala or Nα-Ac-Lys-D-Ala-D-Lac, at its N-terminus to a carboxymethylated dextran-coated layer immobilized on the chip surface by an N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) method as described by our laboratories.22 In addition to its simplicity, this sensor chip design offers the capability to study the primary HB interaction alone because it lacks a secondary binding site such as the penta-glycine crosslink residue or the lipid membrane (Figure 2). The surface density of the D-Ala-D-Ala and D-Ala-D-Lac peptide ligand immobilized in each sensor chip was estimated as ~1.5 × 10−4 and 5.5 × 10−4 nmol peptide/mm2, respectively, from a difference in response units between two flow cells: ∆∆RU = ∆RU (flow cell 1, peptide) – ∆RU (flow cell 2, reference) (Figure S1). Binding In a VAN-susceptible Model. Sensorgrams for VAN binding in the D-Ala-D-Ala model were acquired in a dose-dependent manner at T = 25 °C as shown in Figure 3A. Each shows a trace for drug association and dissociation which demonstrates the specificity of VAN to this cell-wall ligand. Rate constants for the VAN association and dissociation were calculated by the Langmuir analysis for receptor-ligand interaction in a 1:1 stoichiometry (Table 1).27 This led to the determination of values for association rate constant (kon = 3.6 × 105 s−1 M−1) and dissociation rate constant (koff = 7.7 × 10−2 s−1), and as a result, the equilibrium dissociation


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constant KD (= koff/kon) of 2.1 × 10−7 M, which is in agreement with values in literature (KD = 10– 7

– 10–6 M).22,

24, 28

Two unrelated molecules, each of which belongs to a different class of

antibiotics, were tested as negative controls. These include ramoplanin—a cyclic glycolipodepsipeptide that targets Lipid I and II for blocking their conversion and polymerization,29 and daptomycin which binds the lipid membrane in a calcium-dependent manner and causes membrane disruption.30 Each of these did not show any binding response when compared under the identical condition (Figure 3C and 3D). All of these SPR data collected in the D-Ala-D-Ala surface are clearly supportive of the affinity and specificity for VAN binding in this model.


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Biochemistry

Figure 3. Surface plasmon resonance (SPR) sensorgrams from the injection of vancomycin (VAN), oritavancin (ORI) and controls to a VAN-susceptible cell-wall model (A-D) or VANresistant cell-wall model (E-F). Each model surface was made prepared by immobilization of Nα-Ac-Lys-D-Ala-D-Ala (VAN-susceptible) or Nα-Ac-Lys-D-Ala-D-Lac (VAN-resistant) tripeptide molecule on a CM5 sensor chip. These were measured at T = 25 °C, and the concentrations of each antibiotic molecule injected are indicated in each plot. RU = response unit. FC1 = flow cell (peptide). FC2 = flow cell (reference).


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Table 1. Kinetic parameters and KD values of vancomycin (VAN) and oritavancin (ORI) in two cell-wall modelsa

Temp (°C)

D-Alanine-D-Alanine Analyte

VAN 25 ORI

koff (s−1)

kon −1

−1

(s ×M )

D-Alanine-D-Lactate

KD (M)

7.7(±0.6) ×

3.6(±3.1) ×

2.1(±1.8) ×

10−2

105

10−7

3.9(±0.8) ×

4.3(±4.2) ×

10−4

104

kon

koff (s−1)

−1

−1

(s ×M )

KD (M)b 9.1(±0.3)

-

-

8.9(±8.9) ×

1.9(±1.4)

1.2(±1.8)

1.6(±2.7)

10−9

× 10−2

× 104

× 10−6

a

Mean ± SD (N ≥ 5)

b

KD value of VAN in the D-Ala-D-Lac calculated from Scatchard analysis.

× 10−4

ORI was studied for its binding in the same chip at the similar range of concentrations (Figure 3B). Each of its sensorgrams shows a distinct feature in the dissociation phase with an apparently slower dissociation than VAN. Analysis of this slower dissociation led to the value of koff (3.9 × 10−4 s−1), indicating a 200-fold decrease in the dissociation rate relative to VAN. As a result, its KD value of 8.9 × 10−9 M was obtained from the value of kon (4.3 × 104). This suggests that ORI has a greater affinity than VAN (KD = 2.1 × 10−7 M) by a factor of ~24. Our present SPR results are clearly different from those measured in the solution binding study.6 These provide evidence, suggesting that the greater antibacterial activity6,

7

reported against VAN-

susceptible pathogens by ORI might be attributable, in part, to its ability for a tighter cell-wall peptide adhesion, and consequently, more potent inhibition of cell-wall biosynthesis. Binding In a VAN-resistant Model. We performed the SPR study of VAN in the D-Ala-DLac model at T = 25 °C as shown in Figure 3E. As reported,22 only weak responses were detected at ≤30 µM concentrations while increased responses were observed at much higher


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Biochemistry

concentrations. Due to its very fast dissociation, its KD value was estimated by the Scatchard analysis of its dose-response data as 9.1 × 10−4 M (Figure S5). It indicates a large decrease in affinity by three orders of magnitude relative to the D-Ala-D-Ala surface, which is in good agreement with other values reported in literature (KD = 1.5–1.7 × 10−3 M).22,

31

Such loss of

affinity in this cell-wall model validates its resistance to VAN. In contrast, ORI displayed remarkably stronger responses which varied in a dose-dependent manner and are detectable even at low µM concentrations (Figure 3F). Its value of KD was calculated as 1.62 × 10−6 M from its two rate constants (kon = 1.2 × 104 s−1 M−1; koff = 1.9 × 10−2 s−1). Thus ORI retains a significant affinity to the D-Ala-D-Lac peptide which is greater than that of VAN by approximately 3 orders of magnitude. The kinetic basis for this affinity by ORI is due to its slower dissociation than VAN, though its value (koff = 1.9 × 10−2 s−1) represents a ~50-fold increase compared to its koff value in the D-Ala-D-Ala surface. Despite such an increased rate of dissociation (koff), ORI bound in the VAN-resistant surface dissociates as slowly as does VAN bound in the VAN-susceptible surface. Our KD value for ORI determined in the cell wall model is approximately 10-fold higher than its KD (1.05 × 10−7 M)7 measured with lipid II (D-Lac) in liposomes. Such higher affinity by Műnch, et al.7 might be attributable, in part, to contribution by additional non-H bond interactions such as the membrane anchoring effect.6,

17, 21

Our result

suggests that in order to retain its µM affinity to this VAN-resistant ligand, ORI should generate a favorable enthalpy of binding which is sufficient enough to compensate for the repulsive force from the loss of one H bond interaction with the D-Ala-D-Lac peptide ligand.


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(A)

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epi-vancosamine

7.4 A

10.1 A

4'-Chlorobiphenyl Ala- Glu-Lys-D-Ala-D-Ala

(B)

VAN-susceptible cell wall

ORI ORI

PG

PG

PG

PG

PG

VAN-resistant cell wall

D-Ala

D-Lac

ORI

PG

Figure 4. (A) An NMR structure determined for chloroeremomycin in complex with a cell-wall peptide Ala-γGlu-Lys-D-Ala-D-Ala (PDB code 1GAC).32 (B) A three-dimensional model for oritavancin (ORI, left), and a functional role of its 4′-chlorobiphenyl moiety proposed as a motif for inducing a C-H···π or hydrophobic interaction33 with the methyl side chain of the cell-wall peptide. PG = peptidoglycan A Proposed Model for 4′-Chlorobiphenyl. As an approach to understand the molecular basis of the higher affinity by ORI than VAN in each cell-wall model, we propose a possible working model for the role of 4′-chlorobiphenyl moiety in the ORI-peptide interaction as illustrated in Figure 4. The primary HB interaction in this model is based on an existing NMR structure reported by Kline, A, et al.32 for a complex formed between a cell-wall precursor peptide and chloroeremomycin, another glycopeptide antibiotic member which shares an


12

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Biochemistry

identical structure with ORI except the 4′-chlorobiphenyl moiety which is absent. This complex structure offers a clear view on the conformation and orientation for the cell-wall peptide fragment and the drug molecule engaged in their HB interaction. The through-space distance between the nitrogen atom at the epi-vancosamine and the methyl side chain of the terminal DAla is estimated to be 7.4 Å. This distance can be proximal enough for making an interaction with the rigid 4′-chlorobiphenyl moiety (10.1 Å in length) if it is assumed to be attached covalently to the epi-vancosamine nitrogen as seen in ORI. Based on this model, we hypothesize that the chlorine-substituted biphenyl moiety may serve as a π acceptor to engage in an attractive C-H···π interaction33 or hydrophobic interaction with the methyl side chain, contributing to augmenting the preformed HB interaction. Such contribution is believed to play a more critical role in the VAN-resistant cell-wall peptide in which its HB interaction is significantly weakened by the repulsive force from the terminal D-Lac. Competitive Blocking of 4′-Chlorobiphenyl. Of approaches to be considered for interrogating the C-H···π interaction proposed for 4′-chlorobiphenyl, we preferred a ligand blocking experiment because it allows to selectively perturb the interaction without any modification in the aromatic structure. We tested the above hypothesis for ORI by performing a ligand competition experiment in which the contribution of its biphenyl moiety could be selectively blocked by co-injecting a competitive ligand. The ligand, (2-hydroxypropyl)-βcyclodextrin (β-CD),25,

34

was used because its doughnut-shaped structure presents a

hydrophobic cavity with a dimension of ~6 Å in short diameter and ~8 Å in depth, which forms a stable inclusion complex with a range of hydrophobic guest molecules, including biphenylcontaining molecules (KD ~ 10−3 – 10−5 M).35


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Figure 5. Competitive binding experiments of VAN and ORI with (2-hydroxypropyl)-βcyclodextrin (β-CD) at T = 25 °C. (A–C) SPR sensorgrams from the injection of drug alone or the mixture of the drug + β-CD in either the VAN-susceptible or VAN-resistant surface. (D) Plot of fractional inhibition of binding response (F = 1− RU[β−CD]/RU[β-CD]=0) as the function of β-CD concentration co-injected in each surface (mean ± SD; N = 3). Two error bars for ORI are too small and invisible.

The competitive ligand displacement was performed by the injection of VAN or ORI, each incubated with an excess of β-CD mixed with a molar ratio of 1–1000 as shown in Figure 5. In the D-Ala-D-Ala surface, the addition of β-CD made almost no impact on the binding response


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Biochemistry

(∆RU) of VAN. In contrast, the addition of β-CD to ORI led to a decrease in the response which varied in a dose-dependent manner. These results suggest the occurrence of a specific interaction of β-CD with the 4′-chlorobiphenyl moiety in ORI. However, it is notable that the binding was only partially decreased even at a 1000-fold higher amount of β-CD (0.5 mM) where ~74(±12)% of response at equilibrium (prior to dissociation) still retained. The kinetic analysis of the bound species shows the same slow rate of dissociation, and only less than 2-fold decrease in affinity (KD = 16 nM; Table S1). This suggests that the HB network with the D-Ala-D-Ala peptide remains strong enough to keep the biphenyl moiety in close proximity to the peptide side chain, and thus free ORI molecules are the species which contributed to the binding. In comparison, such HB network is not likely achieved by its β-CD inclusion form due to the bulky, hydrophilic nature of β-CD (diameter ≥ 6 Å)35 which brings a steric congestion36 and induces an unfavorable conformational perturbation,37 each contributable to faster dissociation. The importance of such conformational integrity was reported by Miller, et al. in which even a small structural variation in the HB backbone of a vancomycin derivative causes a significant perturbation in its conformation which leads to loss of its activity.10 We then performed the same ligand competition experiment in the D-Ala-D-Lac surface in which the primary HB interaction is weakened significantly. As shown in Figure 5C, the coinjection of β-CD led to the inhibition of binding response by ORI. Its ∆RU decreased not only in a dose-dependent manner but also significantly such that approximately 80% of the binding could be inhibited at the highest dose of β-CD (2.5 mM). Similar to the D-Ala-D-Ala surface, the kinetic analysis of bound species in the D-Ala-D-Lac surface shows almost no difference in affinity between the absence and presence of β-CD (Table S1), indicating that free ORI molecules contributed to the binding. These results suggest that the biphenyl moiety plays a


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more critical role in the D-Ala-D-Lac surface for drug binding, and its competitive occupation by β-CD could result in almost a complete loss of drug binding.

Based on the above competition results with β-CD, we evaluated a possible correlation between the β-CD concentration and the fractional inhibition of binding response (F = 1− RU[β−CD]/RU[β-CD]=0). As plotted in Figure 5D, VAN shows a lack of correlation in the D-Ala-DAla surface, which is consistent with the lack of the 4′-chlorobiphenyl moiety in its structure. In contrast, ORI shows a linear correlation in the D-Ala-D-Lac surface greater than in the D-Ala-DAla surface. The inhibition constant Ki for β-CD was determined in the D-Ala-D-Lac surface as 2.3 × 10−5 M from a ligand competition analysis (1/F = 1 + cKi/[β-CD]; c = 1 + [peptide]surface/KD).38 This Ki value is lower than the average affinity of β-CD to aromatic guest molecules (KD ≈ 10−4 M),35 suggesting that even a partial occupation of the biphenyl moiety and/or steric congestion by the bulky, hydrophilic β-CD could be effective in interfering with the drug-peptide interaction in the VAN-resistant surface.


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Biochemistry

(A)

ex

(B)

= 280 nm ORI + -CD

ex

= 280 nm ORI + D-Ala- D-Ala

[ -CD]/[ORI] = 0

[P]/[ORI] = 0

10

50

(C)

(D) ORI + -CD

ORI + D-Ala- D-Ala

Figure 6. Fluorescence titration experiments of oritavancin (ORI) with (2-hydroxypropyl)-βcyclodextrin (β-CD) and Nα-Ac-Lys-D-Ala-D-Ala. (A, B) Overlaid steady-state fluorescence spectra acquired in response to a molar excess of each ligand titrated, and (C, D) plots of relative fluorescence intensity (F/F0). Titration experiments of ORI (5.0 × 10−5 M) in phosphate buffered saline (PBS), pH 7.4 were carried out at an ambient temperature (23 °C) by excitation at 280 nm. Fluorescence Spectroscopy. The interaction of ORI with β-CD was further confirmed by fluorescence spectroscopy. Figure 6 shows overlaid fluorescence spectra of ORI (λex = 280 nm) recorded in the presence of a large excess of β-CD or the Nα-Ac-Lys-D-Ala-D-Ala peptide in solution. Titration of each ligand resulted in a dose-dependent decrease in the emission intensity at 320 nm (4′-chlorobiphenyl)25 and 370 nm (aglycon chromophore). This suggests that


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fluorescence quenching occurs as a result of the interaction with either β-CD or the cell-wall peptide. However, a clear difference was observed between β-CD and the peptide in which β-CD displayed a greater decrease in the fluorescence intensity at 370 nm than 320 nm. We believe that such greater decrease at 370 nm could be caused by the biphenyl moiety which induces changes in its conformation and orientation after its inclusion into the β-CD cavity.35 A Scatchard analysis performed for relative fluorescence (F/F0) at 370 nm suggests that its β-CD inclusion complex has a KD value estimated as 1.36(±0.03) × 10−4 M (Figure S6), which is in close agreement with KD values reported for small phenyl-based guest molecules (~10−4 M).35 This result provides separate evidence confirming the property of the biphenyl moiety to be able to bind β-CD. In addition, it suggests indirectly that the biphenyl moiety has a certain extent of flexibility in its conformation and orientation which is required as a separate binding motif. Temperature-dependent Affinity. The values of SPR dissociation constant (KD) presented above were all measured at T = 25 °C. We further investigated whether ORI consistently displays greater affinity than VAN regardless of the temperature variation. We performed variable-temperature SPR experiments for VAN and ORI at two other temperatures, T = 15 °C and 30 °C. Kinetic parameters and KD values were determined as summarized in Table S1 from sensorgrams acquired in the D-Ala-D-Ala and D-Ala-D-Lac surface, respectively (Figure S3, Figure S4).


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Biochemistry

Figure 7. (A, B) Values of dissociation constant (KD) for VAN and ORI are plotted as a function of flow temperature in each cell-wall model. (C, D) Thermodynamic parameters for the binding of VAN and ORI to cell-wall peptides on the surface that include ∆H, −T∆S and ∆G (T = 298.15 °K). In each drug, the affinity variation occurred in a temperature-dependent manner in which affinity was lower at higher temperature. For example, as the flow temperature is increased from T = 15 °C to T = 30 °C, the KD values of ORI increased 25- and 9-fold in the D-Ala-D-Ala and D-Ala-D-Lac surface, respectively. VAN also showed an identical trend in affinity variation by showing increases in KD values by a factor of 13- and 3-fold, respectively. In each drug, such a


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lower affinity at a higher temperature is attributed to a combination of changes in two rate constants—an increase in dissociation (koff) and a decrease in association (kon). In summary, ORI consistently showed higher affinity than VAN in each of the surfaces with its lower KD value approximately by two or three orders of magnitude. Thermodynamic Basis of Binding. The dependence of affinity on temperature observed for VAN and ORI provides key information on the thermodynamic basis of the drug-peptide ligand association as reported in numerous types of receptor-ligand interaction.39-41 To investigate this aspect, we performed van′t Hoff analysis (Ln(1/KD) = −∆H/RT + ∆S/R) for KD values obtained at three temperatures per drug (Figure S7, Table S1), and calculated changes in enthalpy (∆H), entropy (∆S) and free energy (∆G), each as plotted in Figure 7. In each of the model surface, a main driving force for drug binding (negative ∆G) is attributed to a favorable contribution from enthalpy (negative ∆H) which is large enough to compensate for a concomitant decrease in entropy (negative ∆S). For example, in the D-Ala-D-Ala surface, the ∆G values for VAN and ORI are estimated to be −37 and −47 kJ/mol, respectively, each of which results from the ∆H value of −116(±44) and −159(±21) kJ/mol, respectively. Thus, binding affinity is determined primarily by change in enthalpy (∆H) which is greater for ORI than VAN. The same trend in the enthalpy-entropy compensation is also observed in the D-Ala-D-Lac surface. Thus, the ∆H value (−104±4) for ORI makes a favorable contribution greater than that (−49±16) of VAN, which leads to a more negative ∆G for ORI than VAN. In summary, the thermodynamic basis for the drug-peptide interaction is determined by the gain in enthalpy which is greater than the loss in entropy. However, ORI shows a greater gain in enthalpy than VAN in each of the two model surfaces. We believe that this larger gain in


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enthalpy by ORI might be attributable possibly to its bivalent mechanism of binding by which its aglycon backbone engages in a primary HB interaction while its biphenyl moiety interacts with the hydrophobic side chain from the cell-wall peptide. While the hydrophobic interaction alone can be weak, its combination with the HB interaction is believed to result in a tighter binding by ORI and even an ability to overcome the repulsive force caused by the lactate terminus in the VAN-resistant surface. Conclusion ORI constitutes one of essential antibiotics which are currently used in the clinic for treating serious bacterial infections.11 Since its discovery two decades ago at Lilly,6 ORI has been a subject of strong interest in its mechanisms of action11,

14

and for drug design.4,

8, 9

Here, we

evaluated SPR binding kinetics for VAN and ORI in two model surfaces, each for VAN susceptibility or resistance, and discovered that these two drug molecules are significantly different in the affinity and modes of binding. Our finding indicates that ORI displays two to three orders of magnitude greater affinity than VAN in each surface, and it also retains affinity (KD ~ µM) to the D-Ala-D-Lac peptide to which VAN practically lacks affinity (KD ~ mM). Our result is in good agreement with potent activities displayed by ORI over VAN in cell culture assays against various Gram(+) pathogens including multidrug-resistant ones. 6, 7, 11, 14 Findings of this current study advance our knowledge and present new insights into the mechanisms of action associated with the class of glycopeptide antibiotics. First, an early study performed by affinity capillary electrophoresis in solution suggested almost no difference between VAN and ORI in their affinity to the cell-wall precursor peptide.6 However, such lack of affinity by ORI to the VAN-resistant peptide appears to be inconsistent with other


21

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mechanisms of action by ORI,11, 14 which occur at inner PG targets7, 12, 18, 19 or deep in the cell membrane because each requires drug binding and penetration through the PG layer. Our result shows otherwise higher affinity by ORI in both VAN-susceptible and VAN-resistant surface and provides a biophysical basis for its ability to reach inner targets. Second, this study describes the molecular basis underlying the higher affinity of ORI. By use of β-CD as a competitive ligand, we were able to identify its 4′-biphenyl moiety as a cooperative motif of binding linked to its primary HB interaction. This motif has potential ability to induce a C-H···π33 or hydrophobic interaction, which can contribute to enhancing the enthalpy in the drug-peptide association. Third, this finding offers a novel insight to be applicable for drug design in the class of cellwall targeting antibiotics. Currently, several approaches9 have been reported for lead design and optimization in the VAN class of glycopeptides which include a multivalent design (dimers,8, 28, 42-44

heterodimers,45,

46

polymeric constructs22,

47, 48

), lipidation,6,

10, 49, 50

and multifunctional

activity.51-53 This present study suggests a bivalent motif of cell-wall binding to be considered in the drug design with a focus on hydrophobic side chains in the cell-wall peptide as a potential subsite. Future efforts will be directed towards testing and advancing this new design concept with variation in the structure, orientation and linker distance of the biphenyl-like motif. Experimental Method Surface Plasmon Resonance (SPR) spectroscopy. SPR experiments were performed with a CM5 sensor chip docked in a Biacore X system (GE Healthcare Life Sciences).22, 23 Two model surfaces for VAN-susceptible and VAN-resistant bacterial cell walls were prepared as described22 by the immobilization of either Nα-Ac-Lys-D-Ala-D-Ala (Sigma-Aldrich) or Nα-Ac-


22

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Lys-D-Ala-D-Lac (Bachem) to the carboxymethylated dextran layer (~100 nm) of the sensor chip. This peptide immobilization was performed by injecting each peptide solution (60 mM; 70 µL, pH 8.5) at a flow rate of 20 µL/min to a flow cell (FC1) pretreated with a 1:1 mixture (50 µL) of 0.4 M EDC and 0.1 M NHS. A reference surface (FC2) was also prepared in the same chip in the same way without the peptide injection. Data Collection and Analysis. Each sensorgram was acquired by injecting an analyte solution (20 µL, HBS-EP buffer, pH 7.4) at a flow rate of 20 µL/min. Data were collected until reaching a full baseline dissociation or 600 second after injection. The chip was frequently treated with 10 mM glycine–HCl (pH 2.5) for surface regeneration. For data analysis, each sensorgram in FC1 was corrected to a reference sensorgram in FC2 to account for the contribution from a nonspecific adsorption and the bulk effect associated with changes in refractive index: ∆RU (corrected) = RU (FC1) − RU (FC2). A global fitting analysis was performed for each corrected sensorgram to a Langmuir model,27 and two rate constants were extracted, which include the rate of association (kon) and the rate of dissociation (koff) (Equation 1–3). Each value for equilibrium dissociation constant (KD = koff/kon) is reported as a mean value (N = 4–5 independent curves). ∆RUt = ∆RUeq × () (dissociation phase)

Equation 1

∆RUt = ∆RUeq × (1 − [(× )()] ) (association phase)

Equation 2

kon = (ks – koff)/C (where C refers to the concentration of analyte)

Equation 3

Inhibition Constant of β-CD. SPR sensorgrams were acquired by injecting a solution of ORI mixed with (2-hydroxypropyl)-β-cyclodextrin (β-CD) in a specific ratio as noted in Figure 5.


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Inhibition constant Ki of β-CD was determined according to the competition equation for ligandreceptor interaction (Equation 4 and 5):38

F=1−

F=

RU [β − CD] RU [β - CD ] = 0

Equation 4

[βCD]added

Equation 5

[ P] FC [βCD]added + K i (1 + ) KD

where F is defined as the fractional inhibition of ORI binding by β-CD added, [P]FC refers to the concentration of the immobilized D-Ala-D-Lac peptide in a flow cell (~1.1 × 10−5 M), and KD refers to the dissociation constant of ORI to the peptide (~1.6 × 10−6 M at 25 °C). Fluorescence Spectroscopy. Fluorescence titration experiments were performed in a Fluoromax-2 fluorimeter (Horiba Scientific) at an ambient temperature. In a representative titration experiment with β-CD (Sigma-Aldrich), a solution of ORI (5.03 × 10−5 M, PBS, pH 7.4; 4.0 mL) was placed in a cuvette to which a specific amount of β-CD (2.51 × 10−2 M, PBS, pH 7.4) was titrated incrementally in a ratio as specified in Figure 6. After 1 min of mixing, its fluorescence spectra were acquired with an emission window at 330 nm by excitation at a variable wavelength of 255, 270, and 280 nm. The titration experiment with the D-Ala-D-Ala peptide was performed in a similar manner except using a lower concentration of Nα-Ac-Lys-DAla-D-Ala (5.02 × 10−3 M, PBS, pH 7.4). Supporting Information. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. Copies of SPR sensorgrams, supplementary figures and Table S1 are provided.


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Corresponding Author *E-mail: [email protected] Author Contributions All authors have made contributions and given approval to the final version of the manuscript. Notes. The authors declare no competing financial interest. Acknowledgements. This work was supported in part by the British Council and Department for Business Innovation & Skills through the Global Innovation Initiative (GII 207). References (1) Walsh, C. (2000) Molecular Mechanisms that Confer Antibacterial Drug Resistance. Nature (London, U.K.) 406, 775–781. (2) Butler, M. S., Hansford, K. A., Blaskovich, M. A. T., Halai, R., and Cooper, M. A. (2014) Glycopeptide antibiotics: Back to the future. J. Antibiot. 67, 631–644. (3) Walsh, C. T., Fisher, S. L., Park, I. S., Prahalad, M., and Wu, Z. (1996) Bacterial Resistance to Vancomycin: Five Genes and One Missing Hydrogen Bond Tell the Story. Chem. Biol. (Oxford, U. K.) 3, 21–28. (4) Okano, A., Isley, N. A., and Boger, D. L. (2017) Peripheral modifications of [Ψ[CH2NH]Tpg4]vancomycin with added synergistic mechanisms of action provide durable and potent antibiotics. Proc. Natl. Acad. Sci. U. S. A. 114, E5052–E5061. (5) Patti, G. J., Kim, S. J., and Schaefer, J. (2008) Characterization of the Peptidoglycan of Vancomycin-Susceptible Enterococcus faecium. Biochemistry 47, 8378–8385. (6) Allen, N. E., Letourneau, D. L., and Hobbs, J. N. (1997) The Role of Hydrophobic Side Chains as Determinants of Antibacterial Activity of Semisynthetic Glycopeptide Antibiotics. J. Antibiot. 50, 677–684.


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TOC Graphic


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Oritavancin Retains a High Affinity for a Vancomycin-Resistant Cell

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