Small-Molecule Suppression of β-Lactam Resistance in Multidrug

Aug 19, 2014 - resistance in bacteria have focused on Gram-positive bacteria; however, multidrug-resistant Gram-negative bacteria pose a significant r...
0 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/jmc

Small-Molecule Suppression of β‑Lactam Resistance in MultidrugResistant Gram-Negative Pathogens Christopher M. Brackett,† Roberta J. Melander,† Il Hwan An,† Aparna Krishnamurthy,† Richele J. Thompson,‡ John Cavanagh,‡ and Christian Melander*,† †

Department of Chemistry and ‡Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695, United States S Supporting Information *

ABSTRACT: Recent efforts toward combating antibiotic resistance in bacteria have focused on Gram-positive bacteria; however, multidrug-resistant Gram-negative bacteria pose a significant risk to public health. An orthogonal approach to the development of new antibiotics is to develop adjuvant compounds that enhance the susceptibility of drug-resistant strains of bacteria to currently approved antibiotics. This paper describes the synthesis and biological activity of a library of aryl amide 2-aminoimidazoles based on a lead structure from an initial screen. A small molecule was identified from this library that is capable of lowering the minimum inhibitory concentration of β-lactam antibiotics by up to 64-fold.

■

INTRODUCTION The emergence of resistance to multiple antimicrobial agents by pathogenic bacteria is a considerable global public health threat. The Centers for Disease Control and Prevention (CDC) estimates that over two million people acquire antibiotic resistant infections each year in the United States, and more than 23000 people die as a result.1 In particular, the six pathogens labeled the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) are considered the greatest threat to human health.2 Of these, multidrug-resistant (MDR) Gram-negative bacteria are a particular concern due to the lack of new drugs in development that have activity against Gram-negative pathogens.3 β-Lactam antibiotics, one of the three largest classes of antibiotics,4 are among the most commonly prescribed and well-tolerated antibiotics currently available, representing >50% of the total antibiotic market in 2009 with sales of over U.S. $22 billion globally.5 As with all antibiotics, however, resistance to β-lactams has become a major problem.6 Our laboratory,7 and others,8 have been developing small-molecule adjuvants that suppress resistance to current clinically used antibiotics, including β-lactams, as an alternative strategy to the development of new microbicidal compounds, to which resistance will likely be rapidly acquired. We recently reported the 2-aminoimidazole (2-AI) aryl compound 1, which suppresses resistance to carbapenem antibiotics in K. pneumoniae, including a New-Delhi metallobeta-lactamase (NDM-1) producing strain.7b Following the success of 2-AI 1 in suppressing the resistance of K. pneumoniae to β-lactam antibiotics, we wished to extend this approach to © XXXX American Chemical Society

the additional Gram-negative ESKAPE pathogens, A. baumannii and P. aeruginosa. The CDC estimates that 12000 healthcareassociated Acinetobacter infections occur in the United States each year, of which almost 7000 are multidrug-resistant and account for approximately 500 deaths. Similarly concerning are infections caused by P. aeruginosa, of which more than 6000 of an estimated 51000 that occur each year are multidrug-resistant and result in approximately 400 deaths.1 Compound 1 was initially tested for the ability to suppress carbapenem resistance in a MDR A. baumannii strain obtained from the ATCC (BAA-1605). This strain is resistant to carbapenem antibiotics, exhibiting minimum inhibitory concentrations (MICs) of 32 and 16 μg/mL for meropenem and imipenem, respectively. Unfortunately, in contrast to the βlactam resistance suppression observed against K. pneumoniae and the colistin resistance suppression observed against both K. pneumoniae and A. baumannii,7d compound 1 exhibited only limited β-lactam resistance suppression activity against this bacterium, lowering the MIC of meropenem 4-fold from 32 to 8 μg/mL at a concentration of 30 μM. On the basis of this modest activity, we initiated a program to discover additional 2-AI adjuvants that have the ability to suppress β-lactam resistance against MDR A. baumannii and P. aeruginosa in an effort to identify scaffolds that are more active than compound 1. We report the identification of a new initial lead compound by screening a related series of aryl 2-AIs that possess antibiofilm activity against A. baumannii, P. aeruginosa, and Escherichia coli.9 Subsequent analogue synthesis and Received: July 11, 2014

A

dx.doi.org/10.1021/jm501050e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Table 1. Suppression of Meropenem Resistance in A. baumannii ATCC BAA-1605 by Compound 1 and Lead Aryl Amide 2Aminoimidazoles

Scheme 1. Synthetic Route to First-Generation Halogenated Aryl 2-AI Library 9a−la

a

Reagents and conditions: (a) H2, Pd−C, THF rt,16 h; (b) ROCl, Et3N, CH2Cl2, rt, 1 h; (c) TFA, CH2Cl2 0° to rt (4:1), 16 h; (d) 3 M HCl, MeOH.

based antibiotic adjuvants is to first determine the MIC of each 2-AI alone and then quantify the effect each 2-AI has upon antibiotic susceptibility at sub-MIC levels. Initial screening was performed with 30 2-AI aryl compounds against a representative MDR A. baumannii strain (BAA-1605), first recording the MIC of each compound and then recording the MIC of meropenem in the presence of 30% the MIC of each 2-AI. Several compounds with increased activity relative to compound 1 were identified from this screen and are displayed in Table 1 (complete results from this initial screen are summarized in Table S1, Supporting Information). Com-

biological evaluation were undertaken to develop this class of small molecules and obtain compounds with the required efficacy and physicochemical properties to potentially allow future evaluation in vivo as suppressors of antibiotic resistance in Gram-negative ESKAPE pathogens.

■

RESULTS AND DISCUSSION

Screening of Aryl 2-AI Series for Suppression of Meropenem MIC against A. baumannii. As we have reported previously, our screening approach to identify 2-AIB

dx.doi.org/10.1021/jm501050e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Table 2. Carbapenem Resistance Suppression of A. baumannii ATCC-1605 by Compound 2 and Initial Halogenated Aryl Amide 2-Aminoimidazole Library compound 2 9a 9b 9c 9d 9e 9f 9g 9h 9i 9j 9k 9l

MIC (μM) 200 >200 >200 >200 50 >200 100 >200 >200 >200 100 50 25

concentration tested (μM)

meropenem MIC (μg/mL)

60 60 60 60 15 60 30 60 60 60 30 15 7.5

32 2 8 8 4 4 16 8 16 2 2 4 8 8

fold reduction

imipenem MIC (μg/mL)

fold reduction

16 4 4 8 8 2 4 2 16 16 8 4 4

16 2 8 8 4 2 16 16 16 2 2 2 4 8

8 2 2 4 8 1 1 1 8 8 8 4 2

Table 3. Carbapenem Resistance Suppression of P. aeruginosa PA53 by Compound 2 and Initial Halogenated Aryl Amide 2Aminoimidazole Library compound 2 9a 9b 9c 9d 9e 9f 9g 9h 9i 9j 9k 9l

MIC (μM) 100 200 200 >200 >200 >200 200 >200 >200 >200 200 100 50

concentration tested (μM)

meropenem MIC (μg/mL)

30 60 60 60 60 60 60 60 60 60 60 30 15

64 32 32 32 32 8 32 16 32 32 16 32 8 16

fold reduction

ceftazidime MIC (μg/mL)

fold reduction

2 2 2 2 8 2 4 2 2 4 2 8 4

64 64 64 64 64 4 64 64 64 64 4 64 8 8

1 1 1 1 16 1 1 1 1 16 1 8 8

reported azido aryl 2-aminoimidazole 79 was reduced to the corresponding aniline derivative by hydrogenation over palladium on carbon. This intermediate was then coupled with various halogenated benzoyl chlorides (accessed either from commercial vendors or from conversion from the corresponding benzoic acid derivative with SOCl2) in the presence of triethylamine to deliver aryl amide intermediates 8a−8l. Subsequently, Boc deprotection with TFA, followed by counterion exchange with HCl, afforded a pilot library of firstgeneration halogenated aryl amide 2-AIs 9a−l. Yields for 9a−l starting from intermediate 7 ranged from 26 to 69%; yields for all reactions can be found in the Supporting Information. This approach delivered a diverse array of halogenated aryl amide 2AIs, including monofluoro, monochloro, and monobromo derivatives, tri- and penta-fluoro derivatives, di- and trichloro derivatives, and trifluoromethyl-containing derivatives. Effect of Library on A. baumannii Carbapenem MICs. Compounds 9a−l were initially tested for β-lactam resistance suppression against A. baumannii strain BAA-1605 in the same manner as the original aryl amide library, by first measuring the MIC of each compound and then measuring the MIC of the carbapenem antibiotics imipenem and meropenem in the presence of the compound at 30% the MIC. The results are displayed in Table 2. The library, along with compound 2, was also screened for β-lactam resistance suppression against three

pounds 2 and 3 (which possess a 3,5-difluoro-substituted benzamide attached at the para-position of the phenyl ring, and a 4-pentyl benzamide attached at the para-position of the phenyl ring, respectively) lowered the meropenem MIC 16-fold from 32 to 2 μg/mL at a concentration of 60 μM, whereas compounds 4, 5, and 6 (which possess a m-pentyl amide, a phexyl amide, and a p-cinnamamide group, respectively) lowered the meropenem MIC 8-fold to 4 μg/mL at a concentration of 30 μM (for compounds 4 and 6) and 60 μM (for compound 5). All of these compounds suppress the meropenem MIC below the CLSI breakpoint for meropenem susceptibility for Acinetobacter spp. of ≤4 μg/mL.10 Of these initial hits, compounds 2 and 3 can be accessed via the same synthetic route, and both present a very attractive scaffold for further analogue development. However, as compound 2 emerged as the lead compound from our previous study of inhibiting ESKAPE pathogen biofilm activity9 and the exploration of the effect of the halogenation pattern about the phenyl ring upon antibiotic resistance suppression activity was particularly attractive to us, further structure−activity relationship (SAR) studies were focused on diversifying the halogenated benzene ring of compound 2. These studies are detailed below. Synthesis of Halogenated Aryl 2-AI Series. The library of halogenated aryl 2-AI derivatives was rapidly assembled using the approach outlined in Scheme 1. Briefly, the previously C

dx.doi.org/10.1021/jm501050e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 1. Second-generation halogenated aryl amide 2-aminoimidazole library.

Table 4. Select Antibiotic Resistance Suppression Data for Second-Generation Aryl Amide 2-Aminoimidazole Library against both A. baumannii and P. aeruginosa BAA-1605

PA53

compound

concentration tested (μM)

meropenem MIC (μg/mL)

fold reduction

concentration tested (μM)

meropenem MIC (μg/mL)

fold reduction

9m 9n 9o 9p 9q 9r 9s 9t 9u

60 60 30 60 30 30 60 60 60

32 8 16 0.5 16 8 8 16 16 4

4 2 64 2 4 4 2 2 8

60 60 30 60 15 60 60 60 60

64 64 64 64 64 64 64 64 64 64

1 1 1 1 1 1 1 1 1

additional MDR A. baumannii clinical isolates obtained from the Walter Reed Army Institute of Research (WRAIR) (strains 5075, 3941, and 3942). Several compounds lowered the MICs of both imipenem and meropenem against the four A. baumannii strains (full antibiotic resistance suppression data against the four A. baumannii strains are summarized in Tables S4−S7, Supporting Information). Similar to the activity observed with meropenem, compound 2 also lowered the imipenem MIC against strain BAA-1605, in this case 8-fold from 16 to 2 μg/mL at a concentration of 60 μM, again taking the MIC below the CLSI breakpoint for susceptibility for Acinetobacter spp. (≤4 μg/mL).10 Compound 9d, which is the chloro analogue of compound 2, possessing a 3,5-dichlorosubstituted phenyl ring, exhibited the greatest effect on carbapenem MICs, effecting 8−16-fold reductions for all four stains, including reducing both carbapenem MICs against BAA1605 by 8-fold at a concentration of just 15 μM. We noted that the presence of a trifluoromethyl substituent imparted greater inherent antibiotic activity to the 2-AI scaffold, with compounds 9k and 9l exhibiting MIC values of 7.5−15 μM against strains BAA-1605 and 5075 compared to 50−200 μM for the non-trifluoromethyl-containing analogues. Compounds 9k and 9l exhibited moderate β-lactam resistance suppression activity, lowering MICs 4−16-fold. Other compounds that showed activity against some or all of the strains tested include the 4-bromo and 3-bromo analogues 9h and 9i and the 3,4,5-trifluoro analogue 9j (Table 2). Effect of Library on P. aeruginosa β-Lactam MICs. We next screened the library along with compound 2 against two P. aeruginosa clinical isolates resistant to meropenem and ceftazidime (obtained from the Ernst laboratory at The University of Maryland, Baltimore). The results against PA53 are shown in Table 3 (full antibiotic resistance suppression data

against both P. aeruginosa strains are summarized in Tables S2 and S3, Supporting Information). Overall, the library was less active against P. aeruginosa than against A. baumannii. Compound 2 was not active against either P. aeruginosa strain at the concentration tested (30 μM). Again, compound 9d was the most active compound, lowering the ceftazidime MIC 16fold from 64 to 4 μg/mL at a concentration of 60 μM against PA53, taking it below the CLSI breakpoint for susceptibility for P. aeruginosa (≤8 μg/mL)m10 and lowering the meropenem MIC 8-fold from 64 to 8 μg/mL. The 3-bromo analogue 9i and the trifluoromethyl-containing analogues 9k and 9l also exhibited some activity against P. aeruginosa, with 9i lowering the ceftazidime MIC against PA53 16-fold to 4 μg/mL at 60 μM. Both 9k and 9l lowered the ceftazidime MIC 8-fold to 8 μg/mL at 30 and 15 μM respectively. Neither 9i nor 9l effected a >4-fold change in meropenem MIC against either strain. Design and Synthesis of Second-Generation Halogenated Aryl 2-AI Library. From the structure−activity data generated from the screening of our initial halogenated aryl amide library, we noted that both halogen identity and placement along the distal aromatic ring had a significant impact on activity. On the basis of this observation, we elected to synthesize a second-generation library in the hope of identifying compounds with greater efficacy than compound 9d. The library was synthesized following the synthetic route used to construct the initial library, as outlined in Scheme 1, and the structures of compounds from the second-generation library are displayed in Figure 1. Given that compound 9d had a 3,5-dichloro substitution pattern, we included a number of additional dichlorobenzamide derivatives with alternative substitution patterns in the secondgeneration library (9m, 9o, and 9p). Due to the activity of the monobromo derivative 9i, and the fact that the dichloro D

dx.doi.org/10.1021/jm501050e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 2. Time kill curve for combinations of 9d and meropenem (MER) against BAA-1605. Solid lines indicate the absence of compound 9d; broken lines indicate the presence of compound 9d (15 μM). Black, control; green, 32 μg/mL MER; yellow, 16 μg/mL MER; red, 4 μg/mL MER; blue, 1 μg/mL MER.

Figure 3. Time kill curve for combinations of 9d and meropenem (MER) against PA53. Solid lines indicate the absence of compound 9d; broken lines indicate the presence of compound 9d (60 μM). Black, control; green, 64 μg/mL MER; yellow, 16 μg/mL MER; red, 4 μg/mL MER; blue, 1 μg/mL MER.

Screening of Second-Generation Library against A. baumannii and P. aeruginosa. The second-generation library was screened for β-lactam resistance suppression against the four A. baumannii strains and two P. aeruginosa strains used to quantify the activity of the initial library, with results from one strain of A. baumannii and P. aeruginosa shown in Table 4 (full antibiotic resistance suppression data summarized in

analogue showed increased activity relative to the monochloro analogue, we synthesized the bromo analogue of compound 9d, compound 9r. To further explore the structure−activity relationship of this class of compound, we also included the mixed-halogen derivatives 9n and 9q and the iodo derivatives 9s, 9t, and 9u. E

dx.doi.org/10.1021/jm501050e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

lead compound, bacterial growth for both species was unaffected when grown with 60 μM 9p. When grown in the presence of both compound 9p and the lowest concentrations of meropenem tested (4 and 1 μg/mL), growth in both strains of bacteria very closely mirrors the control (time kill curves for 9p against BAA-1605 and PA53 can be found in Figures S2 and S3 in addition to tables comparing the reduction in CFUs compared to the control in Tables S10 and S11, Supporting Information). Synergism between 9d and Meropenem against Gram-Negative Bacteria. As another method of verifying that 9d acts synergistically with meropenem against Gramnegative bacteria, we performed checkerboard assays to determine ΣFIC of the combination.11 We chose to work with the same strains as used for the time-dependent study, BAA-1605 and PA53. To obtain a ΣFIC of a combination, the MIC of both compounds must be obtained, and we were unable to determine the MIC of 9d against PA53, as at concentrations above 200 μM 9d was insoluble. However, against BAA-1605, we determined the ΣFIC to be 0.375, indicating synergism between 9d and meropenem.11 Activity of Lead Compound 9d in the Presence of Plasma. To assess the potential of compound 9d under more biomimetic conditions, we investigated the effect of plasma on activity. Accordingly, the MIC of 9d against A. baumannii strain BAA-1605 was measured in 10 and 50% bovine plasma in cation-adjusted Mueller−Hinton broth (CAMHB). In 10% plasma the MIC of 9d was unaffected, remaining at 50 μM (as in CAMHB alone); however, in 50% plasma the MIC of 9d increased to 200 μM, suggesting that the compound exhibits some plasma binding or that some degradation of the compound occurs in plasma. The ability of 9d to suppress meropenem resistance in the presence of plasma was next investigated. At 15 μM (30% MIC) in CAMHB alone 9d lowers the meropenem MIC against BAA-1605 from 32 to 4 μg/mL; however, at the same concentration in 10% plasma, the MIC remained at 32 μg/mL. Increasing the concentration of 9d to 20 and 25 μM resulted in a decrease in meropenem MIC to 16 and 8 μg/mL, respectively (compared to 4 and 2 μg/mL, respectively, in the absence of plasma at this concentration). Further increasing the concentration to 30 μM resulted in a decrease in meropenem MIC to 0.5 μg/mL both in the presence and in the absence of plasma. It can be seen that activity is affected, but not completely eradicated, in the presence of plasma. Hemolytic Activity of Lead Compound 9d. Due to the amphipathic nature of this series of compounds, we wanted to ensure that any compound selected from this series does not affect eukaryotic cell membranes prior to commencing in vivo studies. We therefore first measured the hemolytic activity of compound 9d upon mechanically defibrinated sheep blood as previously reported.7b Compound 9d did not lyse the red blood cells at any concentrations tested (up to 400 μM). Cell Line Toxicity of Lead Compound 9d. To determine whether lead compound 9d exhibits an acceptable toxicity profile to be a viable candidate for in vivo studies, we conducted a methylthiazolyldiphenyl-tetrazolium (MTT) assay, using the HaCaT keratinocyte cell line. We have used this assay previously to establish that related 2-AI antibiofilm compounds do not exhibit toxicity at concentrations used to induce biofilm dispersion/inhibition effects.12 The assay was carried out as previously reported;12 cells were exposed to various concentrations of 9d, and the concentration at which 50% toxicity

Tables S2−S7, Supporting Information). The majority of compounds in this library displayed reduced activity compared to compound 9d, particularly against P. aeruginosa. The dichloro derivative 9o was the only compound that effected any change in the P. aeruginosa MIC, reducing the meropenem MIC against PA54 4-fold from 64 to 16 μg/mL at 30 μM. Compound 9o did not reduce the ceftazidime MIC against this strain, nor did it reduce the MIC of either antibiotic against PA53. The second-generation library did, however, exhibit greater activity against A. baumannii, with some compounds lowering MICs up to 64-fold. Compound 9o displayed potent activity against BAA-1605, suppressing the meropenem MIC 64-fold to 0.5 μg/mL at 30 μM. The iodo derivative 9u reduced meropenem MICs 16-fold against BAA-1605 at 60 μM. From these data, we conclude that replacing the fluoro substituents of the initial hit compound 2 with chloro substituents resulted in an overall increase in activity across the strains, with the 3,5-dichloro derivative 9d exhibiting the most potent resistance suppression activity against both A. baumannii and P. aeruginosa. However, moving from chloro to bromo for the 3,5-disubstituted scaffold did not result in a further increase in activity. Interestingly, altering the substitution pattern of the dichoro analogue from 3,5 to 3,4 resulted in increased activity against one A. baumannii strain, but almost completely abolished activity against P. aeruginosa. Effect of Lead Compound 9d over Time. Time kill curves were constructed to probe the effect of compound 9d on meropenem activity over time. A. baumannii strain BAA-1605 or P. aeruginosa strain PA53 was grown in the presence of 9d, meropenem, or the combination of both, and samples were taken and plated at 2, 4, 6, 8, and 24 h time points (Figures 2 and 3). Compound 9d was used at its active concentration for each bacterial strain (15 μM against BAA-1605 and 60 μM against PA53). Against both bacteria, we observe bacteriostatic growth inhibition at early time points (up to 8 h); however, bacterial growth is similar to that of the control by 24 h. The effect of 9d on bacterial growth in combination with meropenem is most evident at the antibiotic concentrations well below the MIC of the antibiotic alone, 4 and 1 μg/mL (1/8 and 1/32 of the MIC of the antibiotic alone, respectively). For both BAA-1605 and PA53, bacterial growth was unaffected by meropenem alone at 4 or 1 μg/mL; however, there is a marked decrease in the number of CFUs when dosed with 9d. For BAA-1605, at the 6 h time point there is a 95% by LC-MS analysis on a Shimadzu LC-MS 2020 with Kinetex, 2.6 mm, C18 50 × 2.10 mm. 5-(4-Azidophenyl)-2,2-di-tert-butoxycarbonylaminoimidazole-1-carboxylic Acid tert-Butyl Ester (7). The compound was synthesized as previously reported.9 General Synthetic Procedure for Azide Reduction and Amide Coupling. To a solution of anhydrous THF (7.5 mL) and 10% Pd/C (0.015 g) was charged 5-(4-azidophenyl)-2,2-di-tertbutoxycarbonylaminoimidazole-1-carboxylic acid tert-butyl ester (0.3 mmol). Air was removed from the system, and the reaction was back flushed with hydrogen. This process was repeated three times before the reaction was placed under a hydrogen balloon at room temperature for 16 h. After that time, the reaction was filtered to G

dx.doi.org/10.1021/jm501050e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

■

°C with shaking. Samples were taken at 2, 4, 6, 8, and 24 h time points, serially diluted in fresh CAMHB, and plated on nutrient agar. Plates were incubated at 37 °C overnight in stationary conditions, and the number of colonies was enumerated. Checkerboard Assay for FIC Determination. CAMHB was inoculated with BAA-1605 (5 × 105 CFU/mL), and 100 μL was distributed into all wells of a 96-well plate except well 1A. Inoculated CAMHB (200 μL) containing compound (twice the highest concentration tested) was added to well 1A, and 100 μL of the same sample was placed in wells 2A-12A. Column A wells were mixed five times, and 100 μL was transferred to column B. Column B wells were mixed five times before 100 μL was transferred to column C. This procedure was repeated to serially dilute the rest of the columns up to column G (column H was not mixed to allow the MIC of the antibiotic alone to be determined). Inoculated medium (100 μL) containing antibiotic at twice the highest concentration tested was placed in wells A1−H1 and serially diluted in the same manner as before. Row 12 was not mixed in order for the MIC of the compound allowed to be determined. The MICs of both compound and antibiotic alone (from row 12 and column H, respectively) were recorded, as well as for all combinations. The ΣFICs were calculated as follows: ΣFIC = FIC (compound) + FIC (antibiotic), where FIC (compound) is the MIC of the compound in the combination/MIC of the compound alone and FIC (antibiotic) is the MIC of the antibiotic in the combination/MIC of the antibiotic. The combination is considered synergistic when the ΣFIC is ≤0.5, indifferent when the ΣFIC is between 0.5 and 2, and antagonistic when the ΣFIC is ≥2. Hemolysis Assay. Hemolysis assays were performed on mechanically difibrinated sheep blood (Hemostat Laboratories DSB100). Difibrinated blood (1.5 mL) was placed into a microcentrifuge tube and centrifuged for 10 min at 10000 rpm. The supernatant was then removed, and the cells were resuspended in 1 mL of phosphate-buffered saline (PBS). The suspension was centrifuged, the supernatant was removed, and the cells were resuspended two additional times. The final cell suspension was then diluted 10-fold. Test compound solutions were made in PBS in small culture tubes and then added to aliquots of the 10-fold suspension dilution of blood. PBS was used as a negative control and zero hemolysis marker. Triton X (1% sample) was used a positive control (100% lysis marker). Samples were then placed in an incubator at 37 °C while being shaken at 200 rpm for 1 h. Afterward, the samples were transferred to microcentrifuge tubes and centrifuged for 10 min at 10000 rpm. The resulting supernatant was diluted by a factor of 40 in distilled water. The absorbance of the supernatant was then determined with a UV spectrometer at 540 nm. MTT Assay for Cell-Line Toxicity. Human adult, low-calcium, high-temperature (HaCaT) keratinocyte cells were obtained from Dr. Lisa Plano (University of Miami, Miami, FL, USA), who initially obtained the cells from Professor Norbert Fusenig (University of Heidelberg, Heidelberg, Germany).13 Keratinocytes were cultivated in 10% fetal bovine serum−Dulbecco’s modified Eagle’s medium (FBS− DMEM) media (Invitrogen, Carlsbad, CA, USA) with 100 IU/mL of penicillin and 100 μg/mL of streptomycin (Invitrogen) at 37 °C and 5% CO2 until 80−90% confluent. The HaCaT cells were transferred to a 96-well microtiter plate (CellBIND; Corning, Corning, NY, USA) in 100 μL aliquots at a density of 8 × 104 cells/mL in 10% FBS−DMEM complete medium (Invitrogen) and incubated overnight at 37 °C and 5% CO2. Once the cultures were 90% confluent, the compound (9d) was added to the cells using serial dilutions across the plate in duplicate (from 2.5 mM to 1.22 μM). Treated wells and controls (media dilutions, 10% DMSO dilutions, and azide dilutions) were incubated overnight. Using the Roche Cell Proliferation Kit I (Roche Diagnostics Corp., Indianapolis, IN, USA), 10 μL of MTT (5 mg/mL in phosphate-buffered saline; Roche) was added to each well, and the plate was incubated in the dark for 4 h. Next, 100 μL of MTTsolubilizing reagent (10% sodium dodecyl sulfate in 0.01 M HCl; Roche) was added to each well and incubated for 12−18 h. Once the incubation was complete, the plate was scanned at 550 nm, with a reference absorbance of 670 nm, using a TECAN Sunrise plate reader with a rainbow filter (Tecan, Durham, NC, USA).

Article

ASSOCIATED CONTENT

S Supporting Information *

Compound characterization for all novel compounds, 1H and 13 C NMR spectra, and full antibiotic resensitization data. This material is available free of charge via the Internet at http:// pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*(C.M.) E-mail: [email protected]. Phone: (919) 513-2960. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare the following competing financial interest(s): Dr. Melander is a co-founder and a board of directors member of Agile Sciences, a biotechnology company that is seeking to commercialize the antibiofilm and antibiotic sensitization activity of 2-aminoimidazoles.

■

ACKNOWLEDGMENTS We thank Dr. Daniel Zurawski (WRAIR) and Dr. Robert Ernst (University of Maryland, Baltimore) for providing MDR bacterial strains. We thank the DOD DMRDP program (W81XWH-11-2-0115) for their support. The DMRDP program is administered by the Department of Army; U.S. Army Medical Research Acquisition Activity, 820 Chandler Street, Fort Detrick, MD 21702-5014 is the awarding and administering office. The content of this paper does not necessarily reflect the position or the policy of the government, and no official endorsement should be inferred.

■

REFERENCES

(1) CDC. Antibiotic resistance threats in the United States, 2013. (2) Boucher, H. W.; Talbot, G. H.; Bradley, J. S.; Edwards, J. E.; Gilbert, D.; Rice, L. B.; Scheld, M.; Spellberg, B.; Bartlett, J. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin. Infect. Dis. 2009, 48 (1), 1−12. (3) Bassetti, M.; Ginocchio, F.; Mikulska, M. New treatment options against Gram-negative organisms. Crit. Care 2011, 15 (2), 215. (4) Fisher, J. F.; Meroueh, S. O.; Mobashery, S. Bacterial resistance to β-lactam antibiotics: compelling opportunism, compelling opportunity. Chem. Rev. 2005, 105 (2), 395−424. (5) Hamad, B. The antibiotics market. Nat. Rev. Drug Discovery 2010, 9 (9), 675−676. (6) Rice, L. B. Mechanisms of resistance and clinical relevance of resistance to β-lactams, glycopeptides, and fluoroquinolones. Mayo Clin. Proc. 2012, 87 (2), 198−208. (7) (a) Rogers, S. A.; Huigens, R. W., 3rd; Cavanagh, J.; Melander, C. Synergistic effects between conventional antibiotics and 2-aminoimidazole-derived antibiofilm agents. Antimicrob. Agents Chemother. 2010, 54 (5), 2112−2118. (b) Worthington, R. J.; Bunders, C. A.; Reed, C. S.; Melander, C. Small molecule auppression of xarbapenem eesistance in NDM-1 producing Klebsiella pneumoniae. ACS Med. Chem. Lett. 2012, 3 (5), 357−361. (c) Harris, T. L.; Worthington, R. J.; Melander, C. Potent small-molecule suppression of oxacillin resistance in methicillin-resistant Staphylococcus aureus. Angew. Chem. Int. Ed. Engl. 2012, 51 (45), 11254−11257. (d) Harris, T. L.; Worthington, R. J.; Hittle, L. E.; Zurawski, D. V.; Ernst, R. K.; Melander, C. Small molecule downregulation of PmrAB reverses lipid A modification and breaks colistin resistance. ACS Chem. Biol. 2014, 9 (1), 122−127. (8) (a) Taylor, P. L.; Rossi, L.; De Pascale, G.; Wright, G. D. A forward chemical screen identifies antibiotic adjuvants Escherichia coli. H

dx.doi.org/10.1021/jm501050e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

ACS Chem. Biol. 2012, 7 (9), 1547−1555. (b) Podoll, J. D.; Liu, Y.; Chang, L.; Walls, S.; Wang, W.; Wang, X. Bio-inspired synthesis yields a tricyclic indoline that selectively resensitizes methicillin-resistant Staphylococcus aureus (MRSA) to β-lactam antibiotics. Proc. Natl. Acad. Sci. U.S.A. 2013, 110 (39), 15573−15578. (9) Bunders, C. A.; Richards, J. J.; Melander, C. Identification of aryl 2-aminoimidazoles as biofilm inhibitors in gram-negative bacteria. Bioorg. Med. Chem. Lett. 2010, 20 (12), 3797−3800. (10) Clinical and Laboratory Standards Institute Performance Standards for Antimicrobial Susceptibility Testing, 22nd informational supplement M100-S22; Wayne, PA, USA, 2012; Vol. 32, No. 3. (11) Orhan, G.; Bayram, A.; Zer, Y.; Balci, I. Synergy tests by E test and checkerboard methods of antimicrobial combinations against Brucella melitensis. J. Clin Microbiol. 2005, 43 (1), 140−143. (12) Stowe, S. D.; Tucker, A. T.; Thompson, R.; Piper, A.; Richards, J. J.; Rogers, S. A.; Mathies, L. D.; Melander, C.; Cavanagh, J. Evaluation of the toxicity of 2-aminoimidazole antibiofilm agents using both cellular and model organism systems. Drug Chem. Toxicol 35 (3), 310−315. (13) Boukamp, P.; Petrussevska, R. T.; Breitkreutz, D.; Hornung, J.; Markham, A.; Fusenig, N. E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J. Cell Biol. 1988, 106 (3), 761−771.

I

dx.doi.org/10.1021/jm501050e | J. Med. Chem. XXXX, XXX, XXX−XXX