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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 R. Shome, and Jayanta Haldar ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00011 • Publication Date (Web): 04 May 2018 Downloaded from http://pubs.acs.org on May 6, 2018

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ACS Infectious Diseases

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*,† †

Antimicrobial Research Laboratory, New Chemistry Unit, Jawaharlal Nehru Centre for

Advanced Scientific Research, Jakkur, Bengaluru 560064, Karnataka, India. ‡

National

Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI)

Yelahanka, Bengaluru 560064, Karnataka, India.

Corresponding Author E-mail: [email protected]

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-βlactamase (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

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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


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Multidrug-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 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 towards 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 β-lactam ring by bacterial β-lactamase enzymes.10,11 β-Lactamases have been classified into four classes based on their structure and substrate selectivity. Classes A, C and D are ‘serine’ β-lactamases (SBLs) and their mechanism of action is quite similar to the PBPs.12,13 Class B β-lactamases are metallo-βlactamases that comprise zinc (II) in the active centre, 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 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 βlactams, 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 blaNDM-1 gene are also aztreonam-resistant, probably by a different resistance


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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 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 peptideconjugated 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 vancomycin-resistant bacteria.35 In the present study, we rationalized that if this molecule (Dipi-van) can to 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, 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 towards 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. RESULTS AND DISCUSSION


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Synthesis and characterization: Dipicolyl-vancomycin conjugate (Fig. 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 ºC. Then the dipicolyl-1,6hexadiamine (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 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 (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 NDM-1 gene (475 bp). The data confirms the presence of 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. (Supplementary Fig. 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, 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 multi-drug resistance in carbapenem-resistant bacteria. Only the lastline antibiotics, tigecycline (MIC = 0.5-1 µg/mL) and colistin (MIC = 0.5-1 µg/mL) were active


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against these isolates (Supplementary Table S1). The antibacterial activity of dipicolylvancomycin 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). The combined activity (synergistic effect) of meropenem and Dipi-van was evaluated against all the four isolates using checkerboard assay. If combined fractional inhibitory concentration (FIC) of both the agents (the FIC Index; FICI) is ≤0.5, and then such combination is defined 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 NDM-1 isolates to meropenem. According to CLSI (Clinical & Laboratory Standards Institute) guidelines, the susceptibility 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 (>40-fold). Over all, Dipi-van showed synergistic and resensitization profiles with meropenem against all tested four NDM-1 expressing clinical isolates at low concentrations (Table 1).


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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 (Supplementary 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 meropenem-resistant non-metallo-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 (Fig. 2a). This observation reveals that Dipi-van restored meropenem activity against NDM-1 expressing pathogens specifically (Fig. 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 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, Dipi-van was not able to lower the MIC of meropenem (MIC >100 µg/mL) (Fig. 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 Dipivan-Zn2+ complex, due to which Dipi-van could not then interact with the Zn2+ of the NDM-1


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enzyme and was not able to resensitize NDM-1 pathogens to meropenem. The Dipi-van-Zn+2 complex formation was characterized by mass spectrometry (Supplementary Fig. 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, 1-N-phenyl-naphthylamine (NPN) was used as a probe to measure the differential OM penetrating abilities of these compounds. NPN fluoresces strongly in a lipophilic environment such as the vicinity of bacterial membrane. The results indicate that treatment with Dipi and Dipi-van produced a time-dependent increase in fluorescence intensity due to an enhanced membrane permeabilization of bacteria and resultant internalization of NPN. The increase in fluorescence intensity was rapid and significantly high whereas vancomycin was ineffective in showing any increase in fluorescence intensity (Fig. 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 ~ 8.039 and the nitrogens of Dipi-van are available to interact with the Zn2+ of the NDM-1 enzyme. Over-expression of NDM-1and 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


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tag-NDM-1 protein was over-expressed in soluble form and isolated to 95% purity as demonstrated by the SDS-PAGE (Fig. 3a & 3b). Dipi-van inhibits the NDM-1 enzyme activity: To confirm the effect of Dipi-van on NDM-1 activity, we performed NDM-1 inhibition assay in presence of test inhibitors (Dipi-van, Dipi and vancomycin) using nitrocefin as a substrate. Our results demonstrate that Dipi-van and Dipi demonstrated a potent concentration-dependent inhibition of NDM-1 activity in-vitro (Fig. 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 centres, Zn2+ ions and deplete the NDM-1 metal centre Zn2+ ion (Fig. 3d) thereby inactivating the NDM-1 activity. Although Dipi inhibited the NDM-1 enzymatic activity in-vitro but 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 non-specificity, 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 centre of the enzyme. A solution of Dipi-van and NDM-1 enzyme was incubated at 37 ºC for 2 h and then analyzed by HR-MS. The presence of Dipi-van-


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Zn2+ complex was observed, confirming the ability of Dipi-van to deplete the Zn2+from NDM-1 (Supplementary Fig. 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 NDM-1 inhibitor, we studied the in-vitro toxicity of Dipi-van towards mammalian cells (RAW macrophages). Dipi-van did not exhibit any noticeable toxicity towards 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, Dipi-van 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 NDM1 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 sepsis infection model. Sepsis represents a serious clinical condition of a patient with severe infection in all the vital organs which eventually lead 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 h and 24 h post-


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infection 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 post-infection, 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, Fig. 4a) compared to kidneys (~6 log10 CFU/g, Fig. 4b), lungs (~6 log10 CFU/g, Fig. 4c) and spleen (~6 log10 CFU/g, Fig. 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 (Fig. 4). Significantly, this combination therapy was as good as treatment with colistin (Fig. 4).

CONCLUSIONS New Delhi metallo-β-lactamase-1 (NDM-1) conferring Gram-negative bacterial pathogens have emerged globally and resulting in significant human morbidity and mortality. Therefore, it is essential to develop an effective and safe inhibitor of the NDM-1 enzyme. A dipicolylvancomycin (Dipi-van) conjugate overcomes the resistance conferred by NDM-1 and reinstates the activity of carbapenem against carbapenem-resistant 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 in


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tackling serine-β-lactamases (Class 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 Gramnegative bacterial clinical isolates. E. coli (ATCC 25922) and K. pneumoniae (ATCC-BAA2146) 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 (six-week old) were used for animal studies weighing 19 to 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 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


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(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; an early denaturation step at 94 ºC for 5 min, followed by 30 cycles of 30s at 95 ºC, 30s at 60 ºC and 30s at 72 ºC, and then a final extension step for 5 min at 72 ºC. 2% Agarose gel having 0.05 mg/L ethidium bromide was used to analyze the PCR products for 1h at 100V in 1X 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 475bp 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 micro-dilution 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 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


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determination of activity in presence of external zinc sulphate (ZnSO4), Dipi-van and ZnSO4 were pre-mixed in equimolar concentrations (100 µM + 100 µM), then used for the checkerboard assay. Outer membrane permeabilization assay: Mid-log 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 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 N-phenylnaphthylamine (NPN, 10 µM) dye was added.38 Now, test compounds (vancomycin, Dipi-van, Dipi) were added and measured the fluorescence intensity (excitation wavelength: 350 nm; emission wavelength: 420 nm) for 15 minutes 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-1Rev (5′-CCCCCAAGCTTTCAGCGCAGCTTGTCG GCCATGC-3′), which incorporated a HindIII restriction site after the blaNDM-1 stop codon (underlined). The conditions for the amplification was 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. Over-expression 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


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NDM-1 enzyme inhibition assay26: Initially, 5 nM concentration of NDM-1 enzyme was complemented 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 micro plate format and recorded the absorbance at 490 nm (absorbance) using TECAN plate reader (Infinite Pro.) at 37 ºC. IC50s 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) complemented 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 measured the absorbance at 490 nm using TECAN plate reader (Infinite Pro.) at 37 ºC. 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 to 24 g (six-week-old) were used for the experiment. Mice were infected with an intraperitoneal administration of meropenemresistant NDM-1 K. pneumoniae R3934 (~106 CFU/mouse) .26 Then, the mice were treated twice at 2 h and 24 h post-infection with a specified i.p. dose of either 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 post-infection, mice were


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euthanized and collected the major organs such as liver, spleen, kidney and lungs to find out the bacterial density. Organs were placed into 10 mL 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 Supporting Information. The Supporting Information (supplementary figures and tables) is available free of charge on the ACS Publications website. 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, minocycline against the NDM-1 clinical isolates; antibacterial activity of meropenem with and without Dipi. AUTHOR INFORMATION Corresponding Author *Phone: (+91) 80-2208-2565. Fax: (+91) 80-2208-2627. E-mail: [email protected] ORCID Venkateswarlu Yarlagadda: 0000-0001-7476-0078 Paramita Sarkar: 0000-0001-6917-3493 Sandip Samaddar: 0000-0002-2653-9050 Goutham B. Manjunath: 0000-0002-2386-0367


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Susweta D. Mitra: 0000-0003-2388-9810 Krishnamoorthy Paramanandham: 0000-0001-7495-1333 Bibek R. Shome: 0000-0003-4741-7076 Jayanta Haldar: 0000-0002-8068-1015 Notes This work was funded by JNCASR, Bangalore. The corresponding author hereby declares that there is no conflict of interest with co-authors, and this work is legally, financially, and technically unencumbered. ACKNOWLEDGEMENTS We thank Prof. C.N.R. Rao (JNCASR) for his constant support and encouragement. V.Y. acknowledges CSIR, India for senior research fellowship. S.S. is grateful to the Sheikh Saqr laboratory at JNCASR for a post-doctoral fellowship. ABBREVIATIONS NDM-1, New Delhi Metallo-β-lactamase-1; NMR, nuclear magnetic resonance spectroscopy; HR-MS, high-resolution mass spectrometry; MIC, minimum inhibitory concentration; NPN, 1N-phenyl-naphthylamine; HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); CFU, colony forming units; DMEM, Dulbecco's Modified Eagle's Medium; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. REFERENCES 1. Taubes,

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FIGURES

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


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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 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.


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Figure 3. (a & b) SDS-PAGE (5 to 12%) gels of NDM-1 purification. (a) Lane 1- Puregene 4 color Prestained protein molecular weight marker; Lane 2 & 3 - boiled cell extract of E. coli transformants before induction; Lane 4 & 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 Ni-NTA affinity chromatography. (c) NDM-1 enzyme inhibition. Dipi-van and Dipi inhibits NDM-1 (IC50 ~6 µM) unlike vancomycin. (d) NDM-1 activity in presence of external Zn2+ ions. NDM-1 activity was restored after the addition of ZnSO4 at molar equivalents to the inhibitor (30 µM).


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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 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 untreated group with probability P < 0.05. The P value is calculated using students T-test.


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TABLES Table 1. Antibacterial efficacy of meropenem with or without Dipi-van, Dipi-van alone and colistin against NDM-1 positive clinical isolates.


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Table of Contents Graphic:


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