Vancomycin Analogue Restores Meropenem Activity against NDM-1

Here, we have rationally developed an inhibitor for the NDM-1 enzyme, .... dissolved in 1:1 dry DMF/DMSO at 0 °C. Then, the dipicolyl-1,6-hexadiamine...
1 downloads 0 Views 2MB Size
Download PDF
Article Cite This: ACS Infect. Dis. 2018, 4, 1093−1101

pubs.acs.org/journal/aidcbc

Vancomycin Analogue Restores Meropenem Activity against NDM‑1 Gram-Negative Pathogens Venkateswarlu Yarlagadda,† Paramita Sarkar,† Sandip Samaddar,† Goutham Belagula Manjunath,† Susweta Das Mitra,‡ Krishnamoorthy Paramanandham,‡ Bibek Ranjan Shome,‡ and Jayanta Haldar*,†

ACS Infect. Dis. 2018.4:1093-1101. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 08/10/18. For personal use only.

†

Antimicrobial Research Laboratory, New Chemistry Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bengaluru, Karnataka 560064, India ‡ National Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI), Yelahanka, Bengaluru, Karnataka 560064, India S Supporting Information *

ABSTRACT: New Delhi metallo-β-lactamase-1 (NDM-1) is the major contributor to the emergence of carbapenem resistance in Gram-negative pathogens (GNPs) and has caused many clinically available β-lactam antibiotics to become obsolete. A clinically approved inhibitor of metallo-β-lactamase (MBL) that could restore the activity of carbapenems against resistant GNPs has not yet been found, making NDM-1 a serious threat to human health. Here, we have rationally developed an inhibitor for the NDM-1 enzyme, which has the ability to penetrate the outer membrane of GNPs and inactivate the enzyme by depleting the metal ion (Zn2+) from the active site. The inhibitor reinstated the activity of meropenem against NDM-1 producing clinical isolates of GNPs like Klebsiella pneumoniae and Escherichia coli. Further, the inhibitor efficiently restored meropenem activity against NDM-1 producing K. pneumoniae in a murine sepsis infection model. These findings demonstrate that a combination of the present inhibitor and meropenem has high potential to be translated clinically to combat carbapenem-resistant GNPs. KEYWORDS: antibiotic resistance, NDM-1 Gram-negative bacteria, glycopeptide antibiotics, vancomycin, meropenem, antibacterial activity “serine” β-lactamases (SBLs), and their mechanism of action is quite similar to the PBPs.12,13 Class B β-lactamases are metalloβ-lactamases that comprise zinc(II) in the active center and are mechanistically different.13,14 The development of numerous inhibitors of the Class A, C, and D β-lactamases has substantially extended the spectrum of activity of β-lactams.15,16 As yet, a clinically approved inhibitor of Class B metallo-β-lactamases (MBLs) has not been found.17 The prevalence of NDM-1 in E. coli and K. pneumoniae highlights the potential risk of MBLs.18,19 NDM-1 is known to deactivate most β-lactams, even the last generations of carbapenems and cephalosporins, which are frequently considered as “last line antibiotics”, with the exception of aztreonam.18,20 However, recent strains that carry the blaNDM‑1 gene are also aztreonam resistant, probably by a different resistance mechanism.21 Significant strategies have been reported in recent years for the development of MBL inhibitors, but none of them have reached the clinic.22,23

M

ultidrug-resistant bacterial infections are on the rise as many traditional antibiotics have been rendered ineffective.1−3 Gram-negative pathogens (GNPs) such as E. coli and K. pneumoniae have been reported to have acquired resistance to most of the commonly used antibiotics.4 Of particular concern are New Delhi metallo-β-lactamase-1 (NDM-1) expressing pathogens, which are not only resistant to carbapenems but also resistant to a range of other antibiotics including colistin and tigecycline.5−7 There is a dearth of antibiotics to treat such multidrug-resistant infections. The presence of the outer membrane in Gram-negative bacteria offers an additional challenge toward the identification of novel antibacterial compounds.8 The β-lactams are the most commonly used class of antibiotics to treat serious infections caused by GNPs.9 They are cell wall biosynthesis inhibitors that react with the nucleophilic serine residue of transpeptidases that are involved in peptidoglycan cross-linking, thereby deactivating them.6,7 Resistance to β-lactams results from the hydrolysis of the βlactam ring by bacterial β-lactamase enzymes.10,11 β-Lactamases have been classified into four classes on the basis of their structure and substrate selectivity. Classes A, C, and D are © 2018 American Chemical Society

Received: January 8, 2018 Published: May 4, 2018 1093

DOI: 10.1021/acsinfecdis.8b00011 ACS Infect. Dis. 2018, 4, 1093−1101

ACS Infectious Diseases

Article

Figure 1. Structures of vancomycin, meropenem, dipicolyl-1,6-hexadiamine (Dipi), and dipicolyl−vancomycin conjugate (Dipi-van).

thereby restoring the activity of meropenem. Due to the bacterial specificity of vancomycin, it was reasoned that a derivative of vancomycin might provide enhanced selectivity toward bacterial cell wall associated metalloenzymes (NDM-1). Indeed, we observed that Dipi-van efficiently restored meropenem activity in murine models of sepsis infection against NDM-1 expressing K. pneumoniae with no observed toxicity.

One efficient strategy to combat resistance is combination therapy with an antibiotic and a potentiator that enhances the antibiotic’s activity by overcoming the bacterial resistance mechanisms.24−29 Cyclic boronates,30 thiol-containing small molecules,25,31 and dipicolinic acid derivatives32 have been shown to inhibit metallo-β-lactamases, restoring the β-lactam activity. Recently, King et al. reported aspergillomarasmine A (AMA), a fungal metabolite that can interact with the zinc ion (Zn2+) of NDM-1 enzyme (IC50 ∼ 4 μM). AMA could thus prevent the inactivation of meropenem, thereby resensitizing the NDM-1 producing Enterobacteria to meropenem.26 Although AMA is currently undergoing preclinical testing, it has been found to inhibit the human angiotensin-converting enzyme.33 More recently, Sully et al. reported an interesting approach of targeting NDM-1 bacteria by silencing the NDM-1 gene using peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO).34 We have previously reported a dipicolyl−vancomycin conjugate (Dipi-van), which has the ability to bind Zn2+ ion and is active against vancomycinresistant bacteria.35 In the present study, we rationalized that, if this molecule (Dipi-van) can penetrate the Gram-negative bacterial outer membrane, then it should be able to deplete the Zn2+ ion from metallo-β-lactamases and inhibit the enzyme,

■

RESULTS AND DISCUSSION

Synthesis and Characterization. Dipicolyl−vancomycin conjugate (Figure 1, Dipi-van, 4) was synthesized as described previously.35 Briefly, a solution of HBTU in DMF was added slowly to vancomycin solution dissolved in 1:1 dry DMF/ DMSO at 0 °C. Then, the dipicolyl-1,6-hexadiamine (Dipi, 3) was added to it, and the reaction mixture was allowed to stir for 12 h at room temperature. Then, reverse phase HPLC was used to purify Dipi-van from the reaction mixture, and it was characterized by 1H NMR, 13C NMR, and HR-MS. Isolation of NDM-1 Expressing Gram-Negative Pathogens. Bacterial clinical isolates that were resistant to meropenem (minimum inhibitory concentration, MIC > 16 μg/mL) were characterized for the presence of NDM-1 gene 1094

DOI: 10.1021/acsinfecdis.8b00011 ACS Infect. Dis. 2018, 4, 1093−1101

ACS Infectious Diseases

Article

breakpoint for carbapenem antibiotics is ≤4 μg/mL.37 The MIC of meropenem has come down to susceptible limits, 1.5− 3.1 μg/mL in the presence of Dipi-van (Table 1). At 28.7 μg/ mL (1/8th of its MIC), Dipi-van brought down the MIC of meropenem to 3.1 μg/mL (>40-fold) against R3934 and R3949. Dipi-van decreased the MIC of meropenem to 1.5 μg/ mL (>80-fold) at 1/8th of its MIC (28.7 μg/mL) against ATCC 2146, while at 57.4 μg/mL (25 μM), Dipi-van resensitized R3336 to meropenem, MIC ∼ 3.1 μg/mL (>40fold). Overall, Dipi-van showed synergistic and resensitization profiles with meropenem against all four tested NDM-1 expressing clinical isolates at low concentrations (Table 1). Then, the antibacterial efficacy of the Zn2+ binding motif, dipicolyl-1,6-hexadiamine (Dipi, 3), alone and in combination with meropenem was evaluated against all four clinical isolates. Dipi was not active even at 75 μg/mL (100 μM). Also, Dipi at 75 μg/mL did not resensitize these NDM-1 producing pathogens to meropenem (Table S2) whereas Dipi-van was able to resensitize meropenem-resistant bacteria (NDM-1 expressing pathogens) to meropenem at low concentrations (Table 1). This implies that Dipi should be chemically conjugated to vancomycin (Dipi-van) to synergize the activity of meropenem against NDM-1 pathogens. Dipi-van Specifically Restores Meropenem Activity against NDM-1 Positive Pathogens. To check whether Dipi-van is only specific against NDM-1 producing pathogens, the MIC of meropenem was evaluated against the meropenemresistant nonmetallo-β-lactamase producing clinical isolate R3421. Against this isolate, Dipi-van did not exhibit any activity at 230 μg/mL. Also, Dipi-van did not reduce the MIC of meropenem against NDM-1 negative pathogen, R3421, even at 115 μg/mL (Figure 2a). This observation reveals that Dipivan restored meropenem activity against NDM-1 expressing pathogens specifically (Figure 2a). To partially prove the proposed hypothesis on how Dipi-van restored meropenem activity, the antibacterial activity of meropenem in combination with Dipi-van was evaluated in the presence of external Zn2+ by using molar equivalents of zinc sulfate (ZnSO4) as an antagonist against R3934. The results demonstrate that, with 12.5 μM concentrations of ZnSO4, Dipivan was not able to lower the MIC of meropenem (MIC > 100 μg/mL) (Figure 2b). Further, Dipi-van at 100 μM (=230 μg/ mL) was unable to reduce the MIC of meropenem (MIC > 100 μg/mL) in the presence of externally provided Zn2+ of 100 μM. This might be credited to the formation of Dipi-van−Zn2+ complex, due to Dipi-van not being able to interact with the Zn2+ of the NDM-1 enzyme and not being able to resensitize NDM-1 pathogens to meropenem. The Dipi-van−Zn 2+ complex formation was characterized by mass spectrometry (Figure S2). Dipi-van Permeabilizes the Outer Membrane of Gram-Negative Pathogens. The outer membrane (OM) of GNPs plays a pivotal role to their inherent resistance; hence, permeabilization of OM is essential for antibacterial agents that target GNPs.38 GNPs are intrinsically resistant to glycopeptide antibiotics such as vancomycin due to the inability of glycopeptides to permeabilize the Gram-negative bacterial OM. The OM permeabilizing ability of compounds vancomycin at 15 μM (22 μg/mL), Dipi-van at 15 μM (34 μg/mL), and Dipi at 30 μM (22 μg/mL) was studied against R3934. Here, 1N-phenyl-naphthylamine (NPN) was used as a probe to measure the differential OM penetrating abilities of these compounds. NPN fluoresces strongly in a lipophilic environ-

(475 bp) using polymerase chain reaction (PCR) and gel electrophoresis. The primers NDM-F (5′-GGG CAG TCG CTT CCA ACG GT-3′) and NDM-R (5′-GTA GTG CTC AGT GTC GGC AT-3′) were used to amplify the NDM-1 gene.36 UV light was used for the visualization of amplified product corresponding to the NDM-1 gene (475 bp). The data confirms the presence of the NDM-1 gene in K. pneumoniae R3934, K. pneumoniae R3949, and E. coli R3336 whereas MDR K. pneumoniae R3421 turned out to be negative. K. pneumoniae (ATCC-BAA-2146) and E. coli ATCC 25922 were used as positive and negative controls, respectively (Figure S1). Dipicolyl−Vancomycin Conjugate Restores Meropenem Activity in Vitro. The MDR strain and the four NDM-1 bacterial isolates are entitled as R3421 and R3336, R3934, R3949, and ATCC2146 respectively. All the isolates were resistant to commonly used antibiotics such as ciprofloxacin, kanamycin, erythromycin, minocycline, and meropenem. For instance, ciprofloxacin, kanamycin, and erythromycin were not active even at the highest concentration tested, 250 μg/mL. This demonstrates the high level multidrug resistance in carbapenem-resistant bacteria. Only the last-line antibiotics, tigecycline (MIC = 0.5−1 μg/mL) and colistin (MIC = 0.5−1 μg/mL), were active against these isolates (Table S1). The antibacterial activity of the dipicolyl−vancomycin conjugate (Dipi-van) was tested against all the isolates and found to be minimal. Dipi-van showed MIC of 230 μg/mL (100 μM) against R3336, R3934, and R3949 whereas it exhibited MIC of 115 μg/mL against ATCC2146 (Table 1). Table 1. Antibacterial Efficacy of Meropenem with or without Dipi-van, Dipi-van Alone, and Colistin against NDM-1 Positive Clinical Isolates minimum inhibitory concentration meropenem (μg/mL) NDM-1 expressing bacteria Klebsiella pneumoniae R3949 Klebsiella pneumoniae R3934 Klebsiella pneumoniae ATCC BAA2146 Escherichia coli R3336

− Dipi-van

+ Dipi-van at 12.5 μM (28.7 μg/mL)

Dipi-van (μg/ mL)

colistin (μg/ mL)

>100

3.1

230

0.5

>100

3.1

230

0.5

>100

1.5

115

0.5

>230

0.5

>100

12

The combined activity (synergistic effect) of meropenem and Dipi-van was evaluated against all four isolates using the checkerboard assay. If combined fractional inhibitory concentration (FIC) of both agents (the FIC Index; FICI) is ≤0.5, then such combination is defined as synergistic. Dipi-van reduced the MIC of meropenem significantly (3.1 μg/mL) against the three clinical isolates, R3949, R3336, and R3934, indicating the synergistic effect (FICI of ≤0.5) of Dipi-van (Table 1). Notably, Dipi-van at 28.7 μg/mL (12.5 μM), which is 1/8th and 1/4th of its MIC, exhibited high synergy (FICI ∼ 0.15) in combination with meropenem against R3336, R3934, R3949, and ATCC2146, respectively (Table 1). Our results demonstrate that Dipi-van was able to resensitize all the tested NDM1 isolates to meropenem. According to CLSI (Clinical & Laboratory Standards Institute) guidelines, the susceptibility 1095

DOI: 10.1021/acsinfecdis.8b00011 ACS Infect. Dis. 2018, 4, 1093−1101

ACS Infectious Diseases

Article

Figure 2. (a) Antibacterial activity of meropenem and Dipi-van against meropenem-resistant NDM-1 positive clinical isolate K. pneumoniae R3934 and meropenem-resistant NDM-1 negative clinical isolate K. pneumoniae R3421. (b) Antibacterial activity of meropenem, combination of meropenem with Dipi-van at 12.5 μM (28.7 μg/mL), and combination of meropenem with Dipi-van at 12.5 μM in the presence of ZnSO4 (at 12.5 μM) against NDM-1 positive clinical isolate K. pneumoniae R3934. (c) Outer membrane permeabilization of vancomycin, Dipi-van at 15 μM (34 μg/ mL), and Dipi at 30 μM (22 μg/mL) against NDM-1 positive clinical isolate K. pneumoniae R3934.

Figure 3. (a, b) SDS-PAGE (5−12%) gels of NDM-1 purification. (a) Lane 1, Puregene 4 color Prestained protein molecular weight marker; Lanes 2 and 3, boiled cell extract of E. coli transformants before induction; Lanes 4 and 5, boiled cell extract of E. coli transformants after a 14 h induction with 0.1 mM IPTG at 18 °C. (b) Lane 1, Puregene 4 color Prestained protein molecular weight marker; Lane 2, Purified His6 tag-NDM-1 after NiNTA affinity chromatography. (c) NDM-1 enzyme inhibition. Dipi-van and Dipi inhibits NDM-1 (IC50 ∼ 6 μM) unlike vancomycin. (d) NDM-1 activity in the presence of external Zn2+ ions. NDM-1 activity was restored after the addition of ZnSO4 at molar equivalents to the inhibitor (30 μM).

time-dependent increase in fluorescence intensity due to an enhanced membrane permeabilization of bacteria and resultant

ment such as the vicinity of the bacterial membrane. The results indicate that treatment with Dipi and Dipi-van produced a 1096

DOI: 10.1021/acsinfecdis.8b00011 ACS Infect. Dis. 2018, 4, 1093−1101

ACS Infectious Diseases

Article

Figure 4. In vivo antibacterial activity of Dipi-van (10 mg/kg), meropenem (10 mg/kg), colistin (5 mg/kg), and combination of meropenem (10 mg/kg) with Dipi-van (10 mg/kg) in a sepsis infection model against NDM-1 positive clinical isolate K. pneumoniae R3934. Bacterial load measured in the liver (a), kidney (b), lungs (c), and spleen (d) after 48 h. The data are presented as mean ± standard deviation, based on values obtained from 5 mice (n = 5). Differences are considered statistically significant from the untreated group with probability P < 0.05. The P value is calculated using students t test.

internalization of NPN. The increase in fluorescence intensity was rapid and significantly high whereas vancomycin was ineffective in showing any increase in fluorescence intensity (Figure 2c). Although the reason for outer membrane permeability of Dipi-van cannot be conclusively deciphered, it is envisioned that the overall positive charge of the Dipi-van increases at the site of infection, where pH would be less than 7.0, and helps in interacting with the more negatively charged OM of bacteria. At the periplasm, where NDM-1 resides, the pH is higher, ∼8.0,39 and the nitrogens of Dipi-van are available to interact with the Zn2+ of the NDM-1 enzyme. Overexpression of NDM-1 and Its Purification. The plasmid pET30a (+) vector was used to clone the blaNDM‑1 gene using competent E. coli BL21 (DE3), controlled by T7 promoter. The protein production was initiated by 0.1 mM IPTG (isopropyl-β-D-thiogalactopyranoside). His6 tag-NDM-1 protein was overexpressed in soluble form and isolated to 95% purity as demonstrated by the SDS-PAGE (Figure 3a,b). Dipi-van Inhibits the NDM-1 Enzyme Activity. To confirm the effect of Dipi-van on NDM-1 activity, we performed the NDM-1 inhibition assay in the presence of test inhibitors (Dipi-van, Dipi, and vancomycin) using nitrocefin as a substrate. Our results show that Dipi-van and Dipi demonstrated a potent concentration-dependent inhibition of NDM-1 activity in vitro (Figure 3c) with the IC50 of ∼6 μM (13.8 μg/mL) whereas vancomycin had no effect on the activity of NDM-1. Further, the enzymatic activity was restored by addition of excess ZnSO4, indicating that Dipi and Dipi-van

interact with the NDM-1 metal centers, Zn2+ ions, and deplete the NDM-1 metal center Zn2+ ion (Figure 3d), thereby inactivating the NDM-1 activity. Although Dipi inhibited the NDM-1 enzymatic activity in vitro, it was ineffective in resensitizing NDM-1 pathogens to meropenem. In contrast, Dipi-van resensitized NDM-1 pathogens to meropenem at low concentrations. The Dipi-van synergism with meropenem is credited to the cell wall specific nature of Dipi-van which results in more accumulation of Dipi-van at the periplasm where NDM-1 enzyme also resides,18 leading to the effective inactivation of the enzyme. On the other hand, as portrayed by our experimental results, Dipi was unable to reinstate the activity of meropenem against bacteria due to its nonspecificity, in spite of inhibiting the NDM-1 enzyme activity in vitro, apparently due to an inability to accumulate at a sufficient concentration at the site of the enzyme. Next, an experiment was performed to determine whether or not Dipi-van has the ability to deplete the Zn2+ from the active center of the enzyme. A solution of Dipi-van and NDM-1 enzyme was incubated at 37 °C for 2 h and then analyzed by HR-MS. The presence of Dipi-van−Zn2+ complex was observed, confirming the ability of Dipi-van to deplete the Zn2+ from NDM-1 (Figure S3). Toxicology of Dipicolyl−Vancomycin Conjugate. With the recent emergence of NDM-1 producing GNPs as a considerable clinical risk, an effective and nontoxic inhibitor of NDM-1 enzyme would significantly benefit the therapeutic armamentarium. To evaluate the safety profile of the present 1097

DOI: 10.1021/acsinfecdis.8b00011 ACS Infect. Dis. 2018, 4, 1093−1101

ACS Infectious Diseases

Article

NDM-1 inhibitor, we studied the in vitro toxicity of Dipi-van toward mammalian cells (RAW macrophages). Dipi-van did not exhibit any noticeable toxicity toward RAW cells even up to 1000 μg/mL (EC50 > 1000 μg/mL) concentration which is many-fold higher than its MIC value. Further, the systemic toxicity of Dipi-van in mouse models was found to be >100 mg/kg, demonstrating the high tolerability of the compound in animals which also suggests that its LD50 value would be >100 mg/kg.35 Therefore, Dipivan can be a safe inhibitor of NDM-1 for the treatment of bacterial infections caused by NDM-1 bacterial pathogens. Dipi-van Restores Meropenem Activity in Sepsis Infection Model. The resensitization of NDM-1 expressing pathogens in vitro to meropenem in combination with Dipi-van prompted us further to examine the efficacy of the combination in vivo. NDM-1 expressing K. pneumoniae is one of the most difficult pathogens to treat and currently widespread in clinical settings.40 Treatment options are limited against these pathogens, and only tigecycline and colistin are the last-line therapies for such carbapenem-resistant infections.41 Here, the synergistic activity of Dipi-van with meropenem was evaluated against NDM-1 positive K. pneumoniae in a sepsis infection model. Sepsis represents a serious clinical condition of a patient with severe infection in all the vital organs which eventually leads to high mortality rate.42 Mice were infected with an IP dose of ∼106 CFU/mouse of K. pneumoniae R3934. Then, mice were injected twice at 2 and 24 h postinfection with monotherapy of meropenem and Dipi-van as well as a combination of Dipi-van with meropenem. Here, saline and colistin were used as negative and positive controls, respectively. After 48 h postinfection, organs such as lungs, liver, spleen, and kidney were collected and processed to find the bacterial density. For saline treated controls, the bacterial count was found to be more in liver (∼7.5 log10 CFU/g, Figure 4a) compared to kidneys (∼6 log10 CFU/g, Figure 4b), lungs (∼6 log10 CFU/g, Figure 4c), and spleen (∼6 log10 CFU/g, Figure 4d). The bacterial load in all the organs was reduced by ∼1.5 log CFU/g with the treatment of Dipi-van alone. Meropenem monotherapy was also able to reduce the bacterial burden by ∼1.5 log CFU/g in liver and kidneys whereas meropenem was ineffective in spleen and lungs. Conversely, the treatment with combination of Dipi-van and meropenem significantly reduced the bacterial density (3−4 log10 CFU/g) in all the organs compared to vehicle-treated (saline) mice (Figure 4). Significantly, this combination therapy was as good as treatment with colistin (Figure 4).

in tackling serine-β-lactamases (Classes A, C, and D) in clinic. Our findings suggest that Dipi-van is an effective inhibitor of the NDM-1 enzyme that could be developed as an antibiotic adjuvant to combat the infections caused by NDM-1 pathogens.

■

METHODS Materials and Instrumentation. Dipi-van was synthesized and purified as described previously.35 6538-UHD Accurate Mass Q-TOF LC-MS instrument was used to obtain highresolution mass spectra (HR-MS). Clinical isolates were collected from Department of Neuromicrobiology, National Institute of Mental Health and Neuro Sciences (NIMHANS), Hosur Road, Bengaluru 560029, India.43 Vitek 2 Compact 60 system (bioMerieux, France) was used for bacterial identification, and Kirby-Bauer disc diffusion assay was used for selecting the carbapenem-resistant Gram-negative bacterial clinical isolates. E. coli (ATCC 25922) and K. pneumoniae (ATCC-BAA-2146) were procured from MTCC (Chandigarh, India) and ATCC (USA), respectively. Culture media and all the antibiotics were purchased from HIMEDIA and Sigma-Aldrich. Eppendorf 5810R centrifuge was used. TECAN (Infinite series, M200 pro) Plate Reader was used to measure absorbance and fluorescence. RAW macrophages (NCCS Pune) were used for cytotoxicity studies. Animals. Specific-pathogen free BALB/c female mice (sixweek old) were used for animal studies weighing 19−24 g, and the infection study was performed at National Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI). As per the standards, the mice were placed in individually ventilated cages (IVC) provided with a controlled environment. The animal experiments were approved by the Institutional Animal Ethics Committee (IAEC) of National Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI), Bengaluru (881/GO/ac/05/CPCSEA), and performed as per the guidelines of Committee for the purpose of Supervision and Experiments on Animals (CPCSEA), Ministry of Environment and Forests, New Delhi. Systemic toxicity study was done at Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR) following institutional ethical guidelines. PCR and Gel Electrophoresis. Conventional polymerase chain reaction (PCR) technique was employed using the primers NDM-F (5′-GGG CAG TCG CTT CCA ACG GT-3′) and NDM-R (5′-GTA GTG CTC AGT GTC GGC AT-3′) (Eurofins Genomics India Pvt. Ltd., Bengaluru) to amplify the NDM-1 gene. The PCR conditions were as follows: an early denaturation step at 94 °C for 5 min, followed by 30 cycles of 30 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C, and then a final extension step for 5 min at 72 °C. 2% agarose gel with 0.05 mg/L ethidium bromide was used to analyze the PCR products for 1 h at 100 V in 1× Tris Acetate EDTA buffer (TAE buffer).36 As a molecular weight marker, a 100 bp DNA ladder was used (SRL Biolit, Mumbai India). The amplified product bands corresponding to 475 bp were detected under UV light for the presence of NDM-1 gene. In Vitro Antibacterial Assays. In vitro antibacterial activity of the test compounds was measured by the broth microdilution method as described in our previously published protocols.44−48 The antibacterial efficacy of the combination, Dipi-van/Dipi, and meropenem was assayed using a checkerboard assay in nutrient broth.38 Initially, a serial 2-fold diluted solution of 25 μL each of test compounds was added into each well of a 96-

■

CONCLUSIONS New Delhi metallo-β-lactamase-1 (NDM-1) conferring Gramnegative bacterial pathogens have emerged globally and resulted in significant human morbidity and mortality. Therefore, it is essential to develop an effective and safe inhibitor of the NDM-1 enzyme. A dipicolyl−vancomycin (Dipi-van) conjugate overcomes the resistance conferred by NDM-1 and reinstates the activity of carbapenem against carbapenemresistant Gram-negative pathogens. Dipi-van exhibits a promising complementary activity against NDM-1 positive bacteria when combined with a carbapenem antibiotic such as meropenem, both in vitro and in vivo. As illustrated by our results, the NDM-1 resistance can be successfully overcome with Dpi-van, and thus, antibiotic activity has been restored. Inhibitor/drug combination therapy has already been successful 1098

DOI: 10.1021/acsinfecdis.8b00011 ACS Infect. Dis. 2018, 4, 1093−1101

ACS Infectious Diseases

Article

well plate and then followed by the addition of 150 μL of bacterial suspension (∼5.0 × 105 CFU/mL). The 96-well plate was then incubated for a period of 24 h at 37 °C, and the O.D. value was recorded at 600 nm. The experiment was performed in triplicates, and the MIC value was a result of two independent experiments. For determination of activity in the presence of external zinc sulfate (ZnSO4), Dipi-van and ZnSO4 were premixed in equimolar concentrations (100 μM + 100 μM) and then used for the checkerboard assay. Outer Membrane Permeabilization Assay. Midlog phase K. pneumoniae R3934 cells were collected by centrifugation and washed with 5 mM HEPES and 5 mM glucose (108 CFU mL−1). Then, the cell pellet was resuspended in a 1:1 solution of HEPES and glucose. Measurements were performed in a 96-well plate containing 0.2 mL of bacterial suspension using a Tecan Plate Reader. To this solution Nphenylnaphthylamine (NPN, 10 μM) dye was added.38 Now, test compounds (vancomycin, Dipi-van, Dipi) were added, and the fluorescence intensity (excitation wavelength: 350 nm; emission wavelength: 420 nm) was measured for 15 min at room temperature. Construction of NDM-1 Plasmid. The blaNDM‑1 gene produced by E. coli R3336 was amplified by PCR with primers NDM-1-Fwd (5′-CCGGAATTCATGGAATTGC CCAATATTATGCACC-3′), which incorporated an EcoRI restriction site at the 5′-end of the gene (underlined), and NDM-1-Rev (5′-CCCCCAAGCTTTCAGCGCAGCTTGTCG GCCATGC3′), which incorporated a HindIII restriction site after the blaNDM‑1 stop codon (underlined). The conditions for the amplification were followed as described previously.36 Then, the amplified product was purified using a Qiagen spin kit and ligated together to yield pET30a−NDM-1. A 2 μL solution of pET30a−NDM-1 was used to transform 100 μL of competent E. coli BL21(DE3) cells (Novagen). Solid Luria-Bertani (LB) agar plates having ampicillin (50 μg/mL), kanamycin (30 μg/ mL), and 50 μM Zn(NO3)2 were used to select the transformants. Overexpression of NDM-1 Soluble Protein and Its Purification. The protocol for overexpression of NDM-1 protein and its purification was followed as described previously.36 NDM-1 Enzyme Inhibition Assay.26 Initially, 5 nM concentration of NDM-1 enzyme was mixed with 20 μM ZnSO4 for 30 min in HEPES buffer (pH ∼ 7.4). Next, the inhibitor was added to the enzyme and incubated for 20 min. Then, the substrate (Nitrocefin, 30 μM) was added. Assays were performed in 96-well microplate format, and the absorbance was recorded at 490 nm using a TECAN plate reader (Infinite Pro.) at 37 °C. IC50’s were calculated from a plot of percent loss of activity versus inhibitor concentration. Next, an experiment was performed by adding Dipi-van (50 μM) to NDM-1 enzyme (20 nM) in HEPES buffer, pH 7.4. The resultant solution was incubated for 2 h at room temperature and then analyzed by HR-MS for the presence of any Dipi-van−Zn2+ complex. Zn2+ Restoration Assay. NDM-1 (5 nM) combined with 20 μM ZnSO4 was incubated with 30 μM Dipi or Dipi-van for 20 min at room temperature. Nitrocefin (30 μM) and ZnSO4 (30 μM) were added to a final volume of 200 μL, and the absorbance was measured at 490 nm using a TECAN plate reader (Infinite Pro.) at 37 °C.

Cytotoxicity Assay. Cytotoxicity of the Dipi-van was assessed against RAW macrophages as per our previously published protocol.49 In Vivo Antibacterial Activity in Murine Infection Models. Sepsis Infection Model. Female BALB/c mice weighing 19−24 g (six-week old) were used for the experiment. Mice were infected with an intraperitoneal administration of meropenem-resistant NDM-1 K. pneumoniae R3934 (∼106 CFU/mouse).26 Then, the mice were treated twice at 2 and 24 h postinfection with a specified i.p. dose of saline, meropenem (10 mg kg−1), Dipi-van (10 mg kg−1), or a combination of Dipi-van (10 mg kg−1) and meropenem (10 mg kg−1). Here, colistin (5 mg kg−1) was used as a positive control. At 48 h postinfection, mice were euthanized, and the major organs such as liver, spleen, kidney, and lungs were collected to find out the bacterial density. Organs were placed into 10 mL of sterile saline on ice and then homogenized. The dilutions of the homogenate were plated onto the agar plates and incubated for 24 h at 37 °C. The bacterial titer was expressed as log10 CFU/g of organ weight.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsinfecdis.8b00011. Characterization of clinical isolates for the presence of NDM-1; HR-MS (ESI) characterization of Dipi-van− Zn2+ complex; HR-MS characterization of Dipi-van− Zn2+ complex after incubation of Dipi-van with NDM-1 enzyme; MIC data of colistin, tigecycline, ciprofloxacin, erythromycin, kanamycin, and minocycline against the NDM-1 clinical isolates; antibacterial activity of meropenem with and without Dipi (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Phone: (+91) 80-2208-2565. Fax: (+91) 80-2208-2627. Email: [email protected]. ORCID

Venkateswarlu Yarlagadda: 0000-0001-7476-0078 Paramita Sarkar: 0000-0001-6917-3493 Sandip Samaddar: 0000-0002-2653-9050 Goutham Belagula Manjunath: 0000-0002-2386-0367 Susweta Das Mitra: 0000-0003-2388-9810 Krishnamoorthy Paramanandham: 0000-0001-7495-1333 Bibek Ranjan Shome: 0000-0003-4741-7076 Jayanta Haldar: 0000-0002-8068-1015 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was funded by JNCASR, Bengaluru. We thank Prof. C.N.R. Rao (JNCASR) for his constant support and encouragement. V.Y. acknowledges CSIR, India, for a senior research fellowship. S.S. is grateful to the Sheikh Saqr laboratory at JNCASR for a postdoctoral fellowship.

■

ABBREVIATIONS NDM-1, New Delhi metallo-β-lactamase-1; NMR, nuclear magnetic resonance spectroscopy; HR-MS, high-resolution 1099

DOI: 10.1021/acsinfecdis.8b00011 ACS Infect. Dis. 2018, 4, 1093−1101

ACS Infectious Diseases

Article

(17) Crowder, M. W., Spencer, J., and Vila, A. J. (2006) Metallo-βlactamases: novel weaponry for antibiotic resistance in bacteria. Acc. Chem. Res. 39, 721−728. (18) González, L. J., Bahr, G., Nakashige, T. G., Nolan, E. M., Bonomo, R. A., and Vila, A. J. (2016) Membrane anchoring stabilizes and favors secretion of New Delhi metallo-β-lactamase. Nat. Chem. Biol. 12, 516−522. (19) Bushnell, G., Mitrani-Gold, F., and Mundy, L. M. (2013) Emergence of New Delhi metallo-β-lactamase type 1-producing enterobacteriaceae and non-enterobacteriaceae: Global case detection and bacterial surveillance. Int. J. Infect. Dis. 17, e325−e333. (20) Bush, K. (2013) Proliferation and significance of clinically relevant β-lactamases. Ann. N. Y. Acad. Sci. 1277, 84−90. (21) Shakil, S., Azhar, E. I., Tabrez, S., Kamal, M. A., Jabir, N. R., Abuzenadah, A. M., Damanhouri, G. A., and Alam, Q. (2011) New Delhi metallo-β-lactamase (NDM-1): An update. J. Chemother. 23, 263−265. (22) Drawz, S. M., Papp-Wallace, K. M., and Bonomo, R. A. (2014) New β-lactamase inhibitors: A therapeutic renaissance in an MDR world. Antimicrob. Agents Chemother. 58, 1835−1846. (23) Fast, W., and Sutton, L. D. (2013) Metallo-β-lactamase: inhibitors and reporter substrates. Biochim. Biophys. Acta, Proteins Proteomics 1834, 1648−1659. (24) Wright, G. D. (2016) Antibiotic adjuvants: rescuing antibiotics from resistance. Trends Microbiol. 24, 862−871. (25) González, M. M., Kosmopoulou, M., Mojica, M. F., Castillo, V., Hinchliffe, P., Pettinati, I., Brem, J., Schofield, C. J., Mahler, G., Bonomo, R. A., Llarrull, L. I., Spencer, J., and Vila, A. J. (2015) Bisthiazolidines: a substrate-mimicking scaffold as an inhibitor of the NDM-1 carbapenemase. ACS Infect. Dis. 1, 544−554. (26) King, A. M., Reid-Yu, S. A., Wang, W., King, D. T., De Pascale, G., Strynadka, N. C., Walsh, T. R., Coombes, B. K., and Wright, G. D. (2014) Aspergillomarasmine A overcomes metallo-beta-lactamase antibiotic resistance. Nature 510, 503−506. (27) Falconer, S. B., Reid-Yu, S. A., King, A. M., Gehrke, S. S., Wang, W., Britten, J. F., Coombes, B. K., Wright, G. D., and Brown, E. D. (2015) Zinc chelation by a small-molecule adjuvant potentiates Meropenem activity in vivo against NDM-1-producing Klebsiella pneumoniae. ACS Infect. Dis. 1, 533−543. (28) Brem, J., van Berkel, S. S., Aik, W., Rydzik, A. M., Avison, M. B., Pettinati, I., Umland, K. D., Kawamura, A., Spencer, J., Claridge, T. D., McDonough, M. A., and Schofield, C. J. (2014) Rhodanine hydrolysis leads to potent thioenolate mediated metallo-β-lactamase inhibition. Nat. Chem. 6, 1084−1090. (29) Brackett, C. M., Melander, R. J., An, I. H., Krishnamurthy, A., Thompson, R. J., Cavanagh, J., and Melander, C. (2014) Small molecule suppression of β-lactam resistance in multi-drug resistant gram-negative pathogens. J. Med. Chem. 57, 7450−7458. (30) Cahill, S. T., Cain, R., Wang, D. Y., Lohans, C. T., Wareham, D. W., Oswin, H. P., Mohammed, J., Spencer, J., Fishwick, C. W. G., McDonough, M. A., Schofield, C. J., and Brem, J. (2017) Cyclic boronates inhibit all classes of β-lactamase. Antimicrob. Agents Chemother. 61, e02260-16. (31) Tehrani, K. H. M. E., and Martin, N. I. (2017) Thiol-containing metallo-β-lactamase inhibitors resensitize resistant Gram-negative bacteria to Meropenem. ACS Infect. Dis. 3, 711−717. (32) Chen, A. Y., Thomas, P. W., Stewart, A. C., Bergstrom, A., Cheng, Z., Miller, C., Bethel, C. R., Marshall, S. H., Credille, C. V., Riley, C. L., Page, R. C., Bonomo, R. A., Crowder, M. W., Tierney, D. L., Fast, W., and Cohen, S. M. (2017) Dipicolinic acid derivatives as inhibitors of New Delhi metallo-β-lactamase-1. J. Med. Chem. 60, 7267−7283. (33) Mikami, Y., and Suzuki, T. (1983) Novel microbial inhibitors of angiotensin-converting enzyme, aspergillomarasmines A and B. Agric. Biol. Chem. 47, 2693−2695. (34) Sully, E. K., Geller, B. L., Li, L., Moody, C. M., Bailey, S. M., Moore, A. L., Wong, M., Nordmann, P., Daly, S. M., Sturge, C. R., and Greenberg, D. E. (2017) Peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) restores carbapenem susceptibility to

mass spectrometry; MIC, minimum inhibitory concentration; NPN, 1-N-phenyl-naphthylamine; HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); CFU, colony forming units; DMEM, Dulbecco’s Modified Eagle’s Medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

■

REFERENCES

(1) Taubes, G. (2008) The bacteria fight back. Science 321, 356−361. (2) Bush, K., Courvalin, P., Dantas, G., Davies, J., Eisenstein, B., Huovinen, P., Jacoby, G. A., Kishony, R., Kreiswirth, B. N., Kutter, E., Lerner, S. A., Levy, S., Lewis, K., Lomovskaya, O., Miller, J. H., Mobashery, S., Piddock, L. J., Projan, S., Thomas, C. M., Tomasz, A. Z., Tulkens, P. M., Walsh, T. R., Watson, J. D., Witkowski, J., Witte, W., Wright, G., Yeh, P., and Zgurskaya, H. I. (2011) Tackling antibiotic resistance. Nat. Rev. Microbiol. 9, 894−896. (3) Piddock, L. J. (2012) The crisis of no new antibiotics-what is the way forward? Lancet Infect. Dis. 12, 249−253. (4) World Health Organization. (2014) Antimicrobial resistance: global report on surveillance, World Health Organization, Geneva, Switzerland, http://www.who.int/drugresistance/documents/ surveillancereport/en/ (accessed 9th May 2014). (5) Arpin, C., Noury, P., Boraud, D., Coulange, L., Manetti, A., Andre, C., M’Zali, F., and Quentin, C. (2012) NDM-1-Producing Klebsiella pneumoniae resistant to colistin in a French community patient without history of foreign travel. Antimicrob. Agents Chemother. 56, 3432−3434. (6) Bush, K., and Jacoby, G. A. (2010) Updated functional classification of β-lactamases. Antimicrob. Agents Chemother. 54, 969− 976. (7) Mattei, P. J., Neves, D., and Dessen, A. (2010) Bridging cell wall biosynthesis and bacterial morphogenesis. Curr. Opin. Struct. Biol. 20, 749−575. (8) Cox, G., and Wright, G. D. (2013) Intrinsic antibiotic resistance: mechanisms, origins, challenges and solutions. Int. J. Med. Microbiol. 303, 287−292. (9) Butler, M. S., Blaskovich, M. A. T., and Cooper, M. A. (2017) Antibiotics in the clinical pipeline in 2015. J. Antibiot. 70, 3−24. (10) Walsh, T. R., Toleman, M. A., Poirel, L., and Nordmann, P. (2005) Metallo-β-lactamases: the quiet before the storm? Clin. Microbiol. Rev. 18, 306−325. (11) Mao, W., Xia, L., and Xie, H. (2017) Detection of carbapenemase-producing organisms with a carbapenem-based fluorogenic probe. Angew. Chem., Int. Ed. 56, 4468−4472. (12) Cornaglia, G., Giamarellou, H., and Rossolini, G. M. (2011) Metallo-β-lactamases: a last frontier for β-lactams? Lancet Infect. Dis. 11, 381−393. (13) Page, M. I., and Badarau, A. (2008) The mechanisms of catalysis by metallo β-lactamases. Bioinorg. Chem. Appl. 2008, 576297. (14) Spencer, J., Read, J., Sessions, R. B., Howell, S., Blackburn, G. M., and Gamblin, S. J. (2005) Antibiotic recognition by binuclear metallo-beta-lactamases revealed by X-ray crystallography. J. Am. Chem. Soc. 127, 14439−14444. (15) Ehmann, D. E., Jahic, C., Ross, P. L., Gu, R. F., Hu, J., DurandReville, T. F., Lahiri, S., Thresher, J., Livchak, S., Gao, N., Palmer, T., Walkup, G. K., and Fisher, S. L. (2013) Kinetics of avibactam inhibition against class A, C, and D β-lactamases. J. Biol. Chem. 288, 27960−27971. (16) Durand-Réville, T. F., Guler, S., Comita-Prevoir, J., Chen, B., Bifulco, N., Huynh, H., Lahiri, S., Shapiro, A. B., McLeod, S. M., Carter, N. M., Moussa, S. H., Velez-Vega, C., Olivier, N. B., McLaughlin, R., Gao, N., Thresher, J., Palmer, T., Andrews, B., Giacobbe, R. A., Newman, J. V., Ehmann, D. E., de Jonge, B., O’Donnell, J., Mueller, J. P., Tommasi, R. A., and Miller, A. A. (2017) ETX2514 is a broad-spectrum β-lactamase inhibitor for the treatment of drug-resistant Gram-negative bacteria including Acinetobacter baumannii. Nat. Microbiol. 2, 17104. 1100

DOI: 10.1021/acsinfecdis.8b00011 ACS Infect. Dis. 2018, 4, 1093−1101

ACS Infectious Diseases

Article

NDM-1-positive pathogens in vitro and in vivo. J. Antimicrob. Chemother. 72, 782−790. (35) Yarlagadda, V., Sarkar, P., Samaddar, S., and Haldar, J. (2016) A vancomycin derivate with a pyrophosphate binding group: a strategy to combat vancomycin-resistant bacteria. Angew. Chem., Int. Ed. 55, 7836−7840. (36) Shen, B., Yu, Y., Chen, H., Cao, X., Lao, X., Fang, Y., Shi, Y., Chen, J., and Zheng, H. (2013) Inhibitor discovery of full-length New Delhi metallo-β-lactamase-1 (NDM-1). PLoS One 8, e62955. (37) Bulik, C. C., Fauntleroy, K. A., Jenkins, S. G., Abuali, M., LaBombardi, V. J., Nicolau, D. P., and Kuti, J. L. (2010) Comparison of Meropenem MICs and susceptibilities for carbapenemaseproducing Klebsiella pneumoniae isolates by various testing methods. J. Clin. Microbiol. 48, 2402−2406. (38) Yarlagadda, V., Manjunath, G. B., Sarkar, P., Akkapeddi, P., Paramanandham, K., Shome, B. R., Ravikumar, R., and Haldar, J. (2016) Glycopeptide antibiotic to overcome the intrinsic resistance of Gram-negative bacteria. ACS Infect. Dis. 2, 132−139. (39) Kim, Y., Cunningham, M. A., Mire, J., Tesar, C., Sacchettini, J., and Joachimiak, A. (2013) NDM-1, the ultimate promiscuous enzyme: substrate recognition and catalytic mechanism. FASEB J. 27, 1917− 1927. (40) Nordmann, P., Cuzon, G., and Naas, T. (2009) The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria. Lancet Infect. Dis. 9, 228−236. (41) Livermore, D. M., Warner, M., Mushtaq, S., Doumith, M., Zhang, J., and Woodford, N. (2011) What remains against carbapenem-resistant Enterobacteriaceae? Evaluation of chloramphenicol, ciprofloxacin, colistin, fosfomycin, minocycline, nitrofurantoin, temocillin and tigecycline. Int. J. Antimicrob. Agents 37, 415−419. (42) Uppu, D. S., Ghosh, C., and Haldar, J. (2015) Surviving sepsis in the era of antibiotic resistance: Are there any alternative approaches to antibiotic therapy? Microb. Pathog. 80, 7−13. (43) Uppu, D. S., Manjunath, G. B., Yarlagadda, V., Kaviyil, J. E., Ravikumar, R., Paramanandham, K., Shome, B. R., and Haldar, J. (2015) Membrane-active macromolecules resensitize NDM-1 gramnegative clinical isolates to tetracycline antibiotics. PLoS One 10, e0119422. (44) Yarlagadda, V., Akkapeddi, P., Manjunath, G. B., and Haldar, J. (2014) Membrane active vancomycin analogues: A strategy to combat bacterial resistance. J. Med. Chem. 57, 4558−4568. (45) Yarlagadda, V., Konai, M. M., Manjunath, G. B., Ghosh, C., and Haldar, J. (2015) Tackling vancomycin-resistant bacteria with lipophilic-vancomycin-carbohydrate conjugates. J. Antibiot. 68, 302− 312. (46) Yarlagadda, V., Sarkar, P., Manjunath, G. B., and Haldar, J. (2015) Lipophilic vancomycin aglycon dimer with high activity against vancomycin-resistant bacteria. Bioorg. Med. Chem. Lett. 25, 5477−5580. (47) Yarlagadda, V., Konai, M. M., Paramanandham, K., Nimita, V. C., Shome, B. R., and Haldar, J. (2015) In-vivo efficacy and pharmacological properties of a novel glycopeptide (YV4465) against vancomycin-intermediate Staphylococcus aureus (VISA). Int. J. Antimicrob. Agents 46, 446−450. (48) Yarlagadda, V., Samaddar, S., Paramanandham, K., Shome, B. R., and Haldar, J. (2015) Membrane disruption and enhanced inhibition of cell wall biosynthesis: A synergistic approach to tackle vancomycinresistant bacteria. Angew. Chem., Int. Ed. 54, 13644−13649. (49) Yarlagadda, V., Samaddar, S., and Haldar, J. (2016) Intracellular activity of a membrane-active glycopeptide antibiotic against methicillin-resistant Staphylococcus aureus infection. J. Glob. Antimicrob. Resist. 5, 71−74.

1101

DOI: 10.1021/acsinfecdis.8b00011 ACS Infect. Dis. 2018, 4, 1093−1101