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Fatty Acid Comprising Lysine Conjugates: Anti-MRSA Agents that Display In-vivo Efficacy by Disrupting Biofilms with no Resistance Development Mohini Mohan Konai, and Jayanta Haldar Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00055 • Publication Date (Web): 22 Feb 2017 Downloaded from http://pubs.acs.org on February 24, 2017

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Bioconjugate Chemistry

Fatty Acid Comprising Lysine Conjugates: Anti-MRSA Agents that Display In-vivo Efficacy by Disrupting Biofilms with no Resistance Development

Mohini M. Konai and Jayanta Haldar*

Chemical Biology and Medicinal Chemistry Laboratory, New Chemistry Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bengaluru 560064, Karnataka, India.

ABSTRACT: Methicillin-resistant Staphylococcus aureus (MRSA) have developed resistance to antibiotics of last resort such as vancomycin, linezolid and daptomycin. Additionally, their biofilm forming capability has set an alarming situation in the treatment of bacterial infections. Herein we report the potency of fatty acid comprising lysine conjugates as novel anti-MRSA agents, which were not only capable of killing growing planktonic MRSA at low concentration (MIC = 3.1-6.3 µg/mL), but also displayed potent activity against non-dividing stationary phase cells. Further, the conjugates eradicated established biofilms of MRSA. The bactericidal activity of D-lysine conjugated tetradecanoyl conjugate (D-LANA-14) is attributed to its membrane disruption against these metabolically distinct cells. In a mouse model of superficial skin-infection D-LANA-14 displayed potent in-vivo anti-MRSA activity (3 and 4 Log reduction at 20 mg/kg and 40 mg/kg respectively) without showing any skintoxicity even at 200 mg/kg of the compound exposure. Additionally, MRSA could not develop resistance against D-LANA-14 even after 18 subsequent passages, whereas the

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topical anti-MRSA antibiotic fusidic acid succumbed to rapid resistance development. Collectively, the results suggested that this new class of membrane targeting conjugates bears immense potential to treat MRSA infections over conventional antibiotic therapy.

KEYWORDS MRSA infections, Membrane active conjugate, Antibiotic resistance, Antibacterial, Antibiofilm

INTRODUCTION In an era where drug-resistant bacteria are emerging rapidly, biofilm associated infections are becoming more difficult to treat with conventional antibiotic therapy.1,2 Specifically, the treatment of infections caused by drug-resistant Staphylococcus. aureus is exacerbated by the rapid increase in such bacterial strains in the microbial world.3-5 The superbug, methicillinresistant S. aureus (MRSA) is notoriously responsible for majority of hospital-acquired (nosocomial) infections and also a greater percentage of community-acquired infections.6,7 Its association with biofilms has further complicated this challenging situation.8,9 MRSAassociated skin and skin-structure related infections are also very often associated with biofilm formation.10 Biofilm formation in skin infections is favoured by higher adhesion and colonizing tendency of bacteria on the skin surface leading to infections of chronic nature.11 Alongside the topical antibiotic fusidic acid, MRSA have already developed high levels of resistance against all classes of anti-MRSA antibiotics (such as mupirocin, vancomycin, clindamycin and linezolid) present in the clinic.12-16 Unfortunately, the resistance against the last resort Gram-positive antibiotic daptomycin is also reported in literature.17 In this present scenario therefore, new anti-MRSA agents with long-lasting efficacy are in high demand to circumvent the challenging problems created by MRSA infections, especially the one which


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associated with biofilm formation. Although several strategies have already been adopted including the nanoparticle based approach to resolve this challenging situation, the success remain elusive.18-24 In the recent past, an enormous amount of effort has been focused on antimicrobial peptides (AMPs) and lipopeptides and their synthetic mimics to achieve the antibacterial agents with long-lasting efficacy.25-39 This class of molecules are also reported to possess antibiofilm activity in addition to potent antibacterial property.40-42 Mechanism of action of this class of molecules is primarily by targeting the bacterial membrane, which makes it difficult for bacteria to develop resistance against them. Mostly, with an overall positive charge in the molecules, they selectively interact with negatively charged bacterial membrane over zwitterionic mammalian cell membrane.43 As an indication of promises of this class of molecules for future antibacterial therapy, the synthetic molecule LTX-10925 and brilacidin26 have already been entered in clinical trials for development as topical antimicrobial agents. However, the limited success of this class of molecules towards clinical translation calls for the development of new class of antibacterial agents. Towards this goal, herein we report the anti-MRSA activity of a new class of membrane targeting molecules. Lysine conjugated aliphatic norspermidine analogues (LANAs) which could target bacterial biofilms were developed. This class of compounds was reported as novel antimicrobial agents, which displayed potent activity against bacteria and Ebola virus.44,45 Motivated by these initial results, here we further investigated this new class of compounds as possible anti-MRSA agents towards tackling infections associated with biofilms. A new synthetic strategy was developed to prepare this class of conjugates. We initially set out to evaluate the antibacterial efficacy against four different MRSA strains including clinical isolates. We also performed time-kill kinetics to assess the bactericidal activity against all the MRSA strains. Activity against metabolically inactive non-dividing



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stationary phase bacteria was also investigated against these MRSA. In order to investigate the membrane targeted mechanism of action, a membrane depolarization assay was performed against the stationary-phase MRSA in addition to the growing planktonic cells. Furthermore, the biofilm disruption capability was evaluated against established biofilms formed by these MRSA strains. Most importantly, biofilm disruption efficacy of the conjugate, D-LANA-14 (Figure 1), was established in a murine model of superficial skininfection. Finally, we evaluated the long-lasting anti-MRSA activity of this compound by determining the propensity of resistance development in two different MRSA strains. The anti-MRSA antibiotic, fusidic acid was used as the comparative agent to gauge the potential of the compounds as future solutions for biofilm associated MRSA infections.

RESULTS AND DISCUSSION Synthesis of the Compounds. Briefly, this class of compounds are conjugates of the amino acid lysine (L or D) and fatty acids (tetradecanoic and hexadecanoic acids), with norspermidine forming the backbone of the molecules (Figure 1). The two terminal primary amine groups of norspermidine were conjugated with lysine and the fatty acids were appended on the secondary amine of norspermidine backbone. The synthesis of lysine conjugated aliphatic norspermidine analogues (LANAs) was achieved in two steps starting from reported precursor amines.47 In the first step of the reaction two N,N´-Di-Boc-L/DLysine were conjugated with the two primary amino groups of the lipidated amines through amide bond formation using HBTU as coupling agent (Scheme 1). In the second step the Boc groups of resulting compounds (1-4) were deprotected using excess of 50% trifluoroacetic acid (TFA) in DCM to obtain the final compounds. All the compounds including the intermediates were characterized by 1H-NMR and HR-MS and the spectra for the final


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Figure 1.

Chemical structures of lysine-conjugated aliphatic norspermidine analogues

(LANAs) with trifluoroacetate counters.

conjugates are provided in the supporting information (Supporting Information Figure S1S8). Based on the stereochemistry of lysine and number of carbon atoms present in the fatty acid, the compounds were named as follows: L-LANA-14 (L-lysine conjugated aliphatic norspermidine analogue bearing tetradecanoyl chain), D-LANA-14 (D-lysine conjugated aliphatic norspermidine analogue bearing tetradecanoyl chain), L-LANA-16 (L-lysine conjugated aliphatic norspermidine analogue bearing hexadecanoyl chain), D-LANA-16 (Dlysine conjugated aliphatic norspermidine analogue bearing hetradecanoyl chain).

Activity against Growing Planktonic MRSA. Antibacterial activity of the conjugates against growing planktonic bacteria was evaluated by determining their MIC (minimum inhibitory concentration) values. All four compounds were tested against four different methicillin resistant S. aureus (MRSA) and one methicillin susceptible S. aureus (MSSA)



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strain. Among these four MRSA strains, three were clinical isolates (obtained from National Institute of Mental Health and Neurosciences, Bangalore) and the remaining strain was a pathogenic methicillin resistant ATCC strain. We included the antibiotic, methicillin in our study to confirm resistance to the drug in all the MRSA strains used. Table 1 suggests a high level of resistance in all MRSA strains, as perceived from their respective MIC values. Briefly, we found that methicillin had a MIC value 0.8-1.6 µg/mL against MSSA, whereas

Scheme 1. Synthesis of conjugates; LANAs.

Table 1. Antibacterial activity against growing planktonic MRSA and cytotoxicity against RAW cells.

MIC (µg/mL) antibacterial agent

EC50 ( µg/mL)

MSSA MTCC737

MRSA ATCC33591

MRSA R3545

MRSA R3889

MRSA R3890

RAW 264.7 TIB-71

L-LANA-14

3.1-6.3

3.1-6.3

3.1-6.3

3.1-6.3

6.3

66

L-LANA-16

3.1-6.3

3.1-6.3

3.1-6.3

3.1-6.3

3.1-6.3

42

D-LANA-14

3.1-6.3

3.1-6.3

3.1-6.3

3.1-6.3

6.3

72

D-LANA-16

3.1-6.3

3.1-6.3

3.1-6.3

3.1-6.3

3.1-6.3

50

Fusidic Acid

≤ 0.1

≤ 0.1

0.1-0.2

0.1-0.2

≤ 0.1

NDa

Methicillin 0.8-1.6 >25 ND stands for “not determined”

12.5-25

12.5-25

> 25

ND

a


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against resistant strains it displayed a value of 12.5-25 µg/mL (MRSA-R3545 and MRSAR3889) and >25 µg/mL (MRSA-ATCC33591 and MRSA-R3890). When we tested our compounds however, we found that they displayed potent activity against all the S. aureus. Against methicillin susceptible strains, we observed an MIC value of 3.1-6.3 µg/mL for all the compounds. The compounds were also found to retain their potent activity against all the methicillin resistant strains as well, with MIC values in the concentration range of 3.1-6.3 µg/mL. The anti-MRSA antibiotic, fusidic acid was also found to display very potent activity against all these growing planktonic MRSA independent of methicillin susceptibility. But, this class of conjugates are advantageous over fusidic acid, as, they were found to retain their activity (MIC = 3.1-6.3 µg/mL) against the fusidic acid resistant MRSA (MIC of FA = 3200 µg/mL) as well, which were generated after 18 subsequent serial passages (Table S1). Combined together, these observations establish that the conjugates are capable of killing S. aureus strains independent of the levels of acquired antibiotic resistance. Toxicity against Mammalian Cells. Preliminary toxicity studies against human erythrocytes (hRBCs) suggested that all the compounds remain non-hemolytic even at much higher concentration than they displayed antibacterial activity.44 L-LANA-14 and D-LANA-14 bearing tetradecanoyl chain were found to have HC50 values (concentration corresponds to 50% hemolysis) of 588 µg/mL and 730 µg/mL respectively, whereas the hexadecanoyl analogues L-LANA-16 and D-LANA-16 displayed comparatively lesser HC50 values of 264 µg/mL and 283 µg/mL respectively.44 Therefore, very high HC50 values of these compounds clearly indicated selective antibacterial activity over mammalian cells. However, the nontoxic nature of the compounds was further investigated by performing cytotoxicity assay against the Row-cell line. As a measure of toxicity, EC50 (concentration corresponds to 50% cell viability) values of all the compounds were determined. Here too, the tetradecanoyl analogue compounds were found to be less toxic compared to hexadecanoyl analogues.



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Compounds, L-LANA-14 and D-LANA-14 displayed EC50 values of 72 µg/mL and 66

µg/mL respectively, whereas L-LANA-16 and D-LANA-16 possessed values of 42 µg/mL and 50 µg/mL respectively (Table 1). Cells treated with D-LANA-14 even at the concentration of 31.3 µg/mL (5-10 folds higher compared to MIC values) were found to retain ~100% cell viability with intact and regular cell morphology like the untreated case (Supporting Information Figure S9).

Time-kill Kinetics against Actively Growing Planktonic MRSA. Having assessed the potency of these compounds against a panel of MRSA followed by evaluating their cytotoxicity, we were now interested at the rate at which they could affect killing. To this end we were prompted to investigate the time-kill kinetics our optimized conjugate, D-LANA-14, against all four MRSA strains at increasing concentrations of MIC, 2×MIC, 4×MIC through to 8×MIC. We also included fusidic acid at a concentration of 8×MIC for a direct comparison. It was found that although fusidic acid was capable of exerting only a bacteriostatic effect against the MRSA strains, D-LANA-14 was bactericidal in action against all four resistant strains (Figure 2). We did observe strain to strain variation in bactericidal activity however; even as D-LANA-14 was able to kill all the MRSA completely (below experimental detection limit) anywhere between 40-240 min. Additionally, the bactericidal killing effect was found to be dependent on concentration of D-LANA-14 used. At its MIC, killing effect of D-LANA-14 was comparatively slower; with a modest 1-1.5 Log reduction in CFU/mL being observed in the MRSA-ATCC33591 and MRSA-R3545 strains within the experimental time period. At 2×MIC, an improved killing rate was noticed with ≥3 Log reduction in MRSA cell viability (bactericidal activity compared with untreated control). It is also noteworthy that cell viability of MRSA-ATCC33591 reduced below the experimental detection limit after 240 min of compound exposure (Figure 2a) even at this relatively low


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concentration. As we increased concentration, we were able to see faster kinetics of killing; at 4×MIC a reduction of ≥4 Log in the viability of all the MRSA strains was observed. The cell

Figure 2. Time kill-kinetics of D-LANA-14 and fusidic acid (FA) against growing planktonic MRSA: ■ Control;

● FA (8×MIC); ▲ D-LANA-14 (1×MIC); ► D-LANA-14

(2×MIC); ▼ D-LANA-14 (4×MIC); ◄ D-LANA-14 (8×MIC). In the figure asterisks (*) indicated complete killing of bacteria. The detection limit of this experiment is 50 CFU/mL. The data plotted are the average of two independent experiments with their standard deviation.

viability of MRSA-R3545 and MRSA-ATCC33591 strains was also reduced below the experimental detection limit at this concentration. At 8×MIC, D-LANA-14 showed improved activity against the other two remaining bacteria, MRSA-R3889 and MRSA-R3890 whose viability was reduced below the detection limit. At this concentration, D-LANA-14 was in



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fact able to kill MRSA-ATCC33591 completely (below detection limit) within 120 min (Figure 2a). Most notably however, D-LANA-14 was able to completely eliminate the MRSA-R3890 bacterial population within 40 min. Taken together, the results above are indicative of the fact that this class of compounds are bactericidal in nature with fast killing kinetics, and is an improvement over FA, which is bacteriostatic with slow killing kinetics even at the higher concentrations tested (8×MIC).

Figure 3. Antibacterial activity of DLANA-14 and fusidic acid (FA), against stationary phase MRSA. (a) MRSA-ATCC33591. (b) MRSA-R3545. (c) MRSA-R3889. (d) MRSA-R3890. In the figure asterisks (*) indicated complete killing of bacteria. The detection limit of this experiment is 50 CFU/mL. The data plotted are the average of two independent experiments with their standard deviation.


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Activity against Non-dividing Stationary Phase MRSA. A major percentage of bacteria in biofilm associated infections are present in slow growing and non-dividing state. A pertinent question in the evaluation of the efficacy of a candidate antibacterial agent therefore, is its potency against such bacterial cells. We thus evaluated the killing efficacy of D-LANA-14 against non-dividing stationary phases of all four MRSA strains. Antibacterial activity at various concentrations such as 5 µg/mL, 10 µg/mL, 20 µg/mL and 40 µg/mL of D-LANA-14 was investigated in comparison to 40 µg/mL of fusidic acid. Collectively, our results suggested that while fusidic acid is completely inactive against stationary phase MRSA, DLANA-14 displayed a concentration dependent killing efficacy (Figure 3). Although reduction in cell viability was less pronounced at lower concentrations of the compound, we observed complete killing (>5 Log reduction) at a concentration of 40 µg/mL. The cell viability of pretreated and untreated control confirms that bacteria remain at the non-dividing stationary phase during the experimental time period. Therefore, being a protein synthesis targeting antibiotic fusidic acid although displayed potent efficacy against growing planktonic MRSA but remains completely ineffective against these non-dividing stationary cells, whereas D-LANA-14, displayed potent activity against these metabolically inactive MRSA (non-dividing stationary phase) in addition to actively growing planktonic cells.

Anti-biofilm Activity. The formation of bacterial biofilms is a major hurdle towards combating bacterial infections. This extends the desired repertoire of a modern antibacterial agent to include collateral antibiofilm properties. Having established potent antibacterial activity of this class of conjugates against both growing planktonic and non-dividing stationary phase MRSA, their biofilm eradicating property was investigated. To this end, we initially evaluated the MBEC (minimum biofilm eradication concentration) values of all four



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compounds against all four strains of MRSA. We also determined the corresponding MBEC values for fusidic acid. Results summarized in table 2 illustrate that MBEC values of conjugates ranged between 125-500 µg/mL whereas fusidic acid displayed a value of >1000 µg/mL against all the MRSA biofilms. We believe that a combination of factors can likely explain the superior efficacy of the conjugates over fusidic acid towards the eradication of biofilms. Firstly, the compounds bear positive charges, which perhaps enable better interaction with the negatively charged biofilm matrix. It is also conceivable that the overall

Table 2. MBEC values of conjugates against various MRSA strains.

MBEC (µg/mL) antibacterial agent

MRSA ATCC 33591

MRSA R3545

MRSA R3889

MRSA R3890

L-LANA-14

125-250

250-500

250-500

250-500

L-LANA-16

125-250

250-500

250-500

250-500

D-LANA-14

250-500

250-500

250-500

250-500

D-LANA-16

250-500

250-500

250-500

250-500

Fusidic Acid

>1000

>1000

>1000

>1000

biofilm disruption by the compounds is then facilitated by their native antibacterial activity against growing planktonic and stationary phase bacteria alike. In contrast, fusidic acid devoid of positive charge and being inactive against non-dividing stationary phase cells is ineffective in disrupting MRSA biofilms as evidenced by the higher MBEC values. We further performed crystal violet staining to quantify the extent of biofilm disruption in three different MRSA strains (MRSA-ATCC33591, MRSA-R3545 and MRSAR3889). Results suggested that D-LANA-14 was effective in disrupting the established


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biofilms of all the MRSA, whereas antibiotic fusidic acid remained ineffective even at higher concentrations (Figure 4). The reduction in biomass was quantified by crystal-violet staining.

Figure 4. Quantification and visualization of biofilm disruption by D-LANA-14: Quantification of biofilm disruption by crystal-violet staining. (a) MRSA-ATCC33591. (b) MRSA-R3545. (c) MRSA-R3889. Percentage biomass remaining after 24 h of compound treatment was calculated by considering the untreated case 100 percentage. The data plotted in the figure is the average of two independent experiments with their standard deviation. (d) Visualization of biofilm disruption formed by MRSA-ATCC33591 through confocal lesser scanning microscopy (CLSM). Contrast and brightness adjusted images are presented here and the scale bar is 10 µm in the figure.



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D-LANA-14 was found to display a dose dependent reduction in bacterial biomass. We were able to observe around 10% reduction in biofilm biomass of MRSA-ATCC33591 when exposed to a 5 µg/mL (~MIC) of D-LANA-14. Similarly, at a concentration of 10 µg/mL (~2×MIC), approximately 15-20% of biomass reduction was observed against all the MRSA strains. However, at the next concentration of 20 µg/ml, D-LANA-14 displayed a strain dependent efficacy. Against the ATCC strain, about 70% reduction in biomass was observed, compared to around 30% and 40% reductions in biomass for the clinically isolated strains MRSA-R-3545 and MRSA-R3889. However, at 40 µg/mL, D-LANA-14 exhibited 70%, 80% and 90% reduction in biomass for MRSA-R-3545, MRSA-R3889 and MRSAATCC33591 respectively. In contrast, the antibiotic fusidic acid could reduce the biomass of MRSA-ATCC33591 and MRSA-R3889 by 10-20% at concentration of 40 µg/mL and no biomass reduction was noticed for the strain MRSA-R3545 (Figure 4). Together with the high MBEC values, this again reinforced the inability of fusidic acid in disrupting MRSA biofilms that consist of metabolically inactive bacterial populations in majority. In order to visualize eradication of MRSA biofilm by D-LANA-14 confocal microscopy was performed. As can be seen from the confocal images (Figure 4d), D-LANA14 could significantly reduce the amount of biomass of an established MRSA ATCC33591 biofilm at 40 µg/mL. The untreated biofilm (control), shows a compact and dense biofilm on the glass coverslip (Figure 4d). When treated with fusidic acid there was no reduction in the biofilms biomass (Figure 4d), which explain its inability in treating biofilm associated MRSA infections. These results confirm that this class compounds are effective in disrupting established biofilms formed by MRSA unlike conventional anti-MRSA antibiotics. Therefore, D-LANA-14 bears significant potential as the therapeutic option in the treatment of MRSA infections associated with biofilms.


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Mechanism of Action. Having established the antibacterial activity against both growing planktonic and non-dividing stationary phase MRSA and their anti-biofilm properties, the membrane targeted mechanism of action of D-LANA-14 was studied by using the membrane potential sensitive dye DISC3(5). Before the addition of compound, the fluorescence intensity remained constant at 100 (Figure 5 and Supporting Information Figure S10). After the addition of compound at 6 min, the fluorescence intensity started increasing which indicated

Figure 5. Membrane depolarization of non-dividing stationary phase MRSA by D-LANA-14. (a) MRSA-ATCC33591. (b) MRSA-R3545. (c) MRSA-R3889. (d) MRSA-R3890. The data demonstrated in the figure are the average of three different experiments and the average of normalized fluorescence intensity with standard deviation is plotted in the figure.



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changes in bacterial membrane potential created by the compound exposure. This shows that D-LANA-14 perturbs the membrane potential of all four MRSA irrespective of actively growing or non-growing cell states. This effect is seen to increase with increasing concentration of D-LANA-14 and we observed the same trend against both metabolically active (planktonic) and metabolically inactive (stationary phase) bacteria. This suggests that membrane disruption by D-LANA-14 results in its efficacy against both growing planktonic and non-dividing stationary phase MRSA alike.

Figure 6. In-vivo activity of D-LANA-14 against murine model of superficial skin-infection. (a) Details of experimental design. (b) Bacterial titre in skin-infection model.

In-vivo Anti-MRSA Activity against Murine Model of Skin-infections. In order to investigate the in-vivo potential of D-LANA-14 as an anti-MRSA agent, its efficacy in a skininfection biofilm model in mice was evaluated. After the establishment of biofilms on the skin of the mice by following our reported protocol,46 we administered a treatment regimen consisting of 4 doses of D-LANA-14 (20 mg/kg and 40 mg/kg) or fusidic acid (40 mg/kg) for 4 consecutive days (24 h dosing interval). The experimental design is demonstrated in figure 6a. D-LANA-14 was able to bring down bacterial burden significantly over that of fusidic


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acid compared to the saline treated controls. A 3 Log and 4 Log reduction in bacterial burden was found at doses of 20 mg/kg and 40 mg/kg of D-LANA-14 respectively (Figure 6b). Treatment with fusidic acid at 40 mg/kg on the other hand, resulted in a reduction of hardly 1 Log. These results illustrated superior potential of D-LANA-14 over fusidic acid, indicating that our in-vitro comparative studies directly translate into in-vivo settings. Given the in-vivo validation, D-LANA-14 and other compounds of this class hold significant promise as a next generation therapeutic modalities in alleviating MRSA infections.

Acute Dermal Toxicity. The acute dermal toxicity was performed in accordance with the OECD guidelines. For the purpose of the study, we used 200 mg/kg of D-LANA-14 (5-10 times the concentration used in the subsequent infection study described above). Visual inspection of mice revealed that the application of the compound caused no adverse effects including irritation, tremors, convulsions, salivation and so on. Although, the detailed toxicity study is still under investigation yet normal fur growth of treated group of mice clearly indicated non-toxic nature of D-LANA-14. Almost complete regeneration of fur after 14 day treatment with D-LANA-14 and in untreated mice without any morbidity further confirmed the non-toxicity of this compound for its topical application (Supporting Information Figure S11).

Propensity to Induce Bacterial Resistance. In the face of a recurrent emergence of drugresistant MRSA, long-lasting antibacterial efficacy is a highly sought after attribute in modern day antibacterial compounds. This prompted us to assess the longevity of D-LANA14 efficacy in the context of resistance development. To this end, we performed resistance development studies against two different MRSA strains. Results suggested that neither MRSA strain was able to develop resistance against this new compound even after 18



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subsequent passages (Figure 7). We observed a negligible increase in the MIC of D-LANA14 during the entire experimental time period. In contrast, the topical antibiotic FA showed a rapid increase in MIC values. In fact fusidic acid, exhibited a ~250-fold and ~500-fold increase in MIC value after only six continuous passages for the MRSA-ATCC and MRSAR3889 strains respectively. These values reached 5000-fold after nine passages for both MRSA strains. After 15 passages, a staggering ~30000-fold increase in MIC was observed, which plateaued for the remainder of the experiment. These results indicate that MRSA strains find difficulty in acquiring resistance against this class of membrane targeting compounds, but rapidly develop resistance against the protein synthesis targeting antibiotic fusidic acid. This long-lasting anti-MRSA efficacy of D-LANA-14 further elucidates the real potential of this class of compounds to be developed as antibacterial therapies of the future.

Figure 7.

Resistance development studies of D-LANA-14 and fusidic acid (FA). (a)

Comparison in fold of MIC increased against MRSA-ATCC33591. (b) Comparison in fold of MIC increased against MRSA-R3889. For each passage, MIC values were determined by visually observing the turbidity. The increase in fold of MIC was determined by dividing MIC values of each passage by the initial MIC value (zero passage).


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CONCLUSIONS MRSA-associated infections are a rapidly growing problem worldwide. Herein we developed a novel class of anti-MRSA agents, LANAs, to combat such bacterial infections. The antiMRSA efficacy of this new class of compounds was compared with the antibiotic fusidic acid; an exceedingly prescribed antibiotic in the treatment of MRSA associated skininfections. This class of compounds are highly bactericidal and inactivate both actively growing planktonic and non-dividing stationary phase MRSA, acting primarily by disrupting the membrane potential. These compounds are also capable to eradicate established biofilms of MRSA, whereas the antibiotic fusidic acid remains completely ineffective. Most importantly, this in-vitro antibacterial activity is retained under in-vivo conditions in a mouse model of skin-infections without signs of acute toxicity. This emphasizes the potency of this new class of compounds as anti-MRSA agents. Additionally, MRSA are unable to develop resistance against D-LANA-14 indicating long-lasting anti-MRSA efficacy of this class of compounds. Collectively all the results suggest that this new class of membrane targeting conjugates possess enormous promise as the therapeutic regimen in curing biofilm-associated MRSA infections.

EXPERIMENTAL SECTION Materials and Instruments:

Organic solvents such as dichloromethane (DCM) and

anhydrous N,N-dimethylformamide (DMF) were purchased from Spectrochem (India) and DCM was dried before its use. Other solvents of LR grade (chloroform and methanol) were obtained from SDFCL (India) and were distilled before their use. L- and D-lysine, Di-tert butyl

dicarbonate

(Boc2O),

N,N,N´,N´-tetramethyl-O-(1H-benzotriazol-1-yl)

uranium

hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIPEA) were purchased from Spectrochem (India). Analytical thin layer chromatography (TLC) was obtained from Merck,



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which is pre-coated with silica gel 60 F254 was used to monitor the reactions. The visualization of TLC plate was performed by using UV or Iodine. Column chromatography was performed on silica gel (60-120 mesh), was obtained from SDFCL (India). Characterization of the compounds was performed by using Nuclear magnetic resonance (NMR) spectroscopy, which were recorded by using Bruker (AV-400) 400 MHz spectrometer in deuterated solvents (CDCl3 or DMSO-d6). The mass spectra of the compounds were obtained by using 6538-UHD Accurate mass Q-TOF LC-MS instrument. The control antibiotics, fusidic acid (Fusidic acid sodium salt-F0881 SIGMA) and methicillin (Methicillin sodium salt-51454 SIGMA) used in the study were obtained from SigmaAldrich. Bacterial growth media (nutrient broth and nutrient agar) were and any other materials for microbiology experiments were obtained from HiMedia. Tecan Infinite Pro series M200 Microplate Reader was used for optical density (O.D.) measurement or fluorescence measurement.

Synthesis and Characterization.

For the present study, this class of compounds was

prepared by following a new synthetic strategy compared to our previously reported synthetic scheme.44 Herein, the synthesis was achieved in two simple reaction steps. The first step of reactions consisting of amide coupling reaction and in the second step deprotection of Bocgroups yielded the final compounds (Scheme 1).

General Procedure for Synthesizing 1-4. At first, 2.4 equivalents of N,N´-Di-Boc-L/DLysine (prepared by following reported protocol)44 were dissolved in dry DCM at 0 ºC. After that 4 equivalents of DIPEA was added followed by 2.4 equivalents of HBTU. Then DMF (1/4 of DCM) was added to the reaction mixture and allowed to stir for 10-15 min. Next, 1 equivalent of precursor amines dissolved in dry DCM and another 2 equivalents of DIPEA


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followed added to the reaction mixture drop wise. The reaction mixture was then allowed to stir for 48h at RT. Reaction solvent was then removed by using rotary evaporator under reduced pressure. After that, the residue was diluted in ethyl acetate and washed with 1N HCl (3 times) followed by saturated Na2CO3 solution (3 times). At the end, the ethyl acetate layer was collected after passing through anhydrous sodium sulphate. Column chromatography on silica gel (60-120 mesh) was then performed by using different ratios of methanol and chloroform as eluent to accomplish the compounds 1-4 with 65-68% yield.

N,N-bis-[{3-(Boc-LLys-Boc)amido}propyl]tetradecanamide (1): Yield-68%;

1

H-NMR

(400 MHz, CDCl3) δ/ppm: 7.328 (s, R-CO-N(-CH2-CH2-CH2-NH-CO-CH(NHBoc)-CH2CH2-CH2-CH2-NHBoc)2,

2H),

5.382-5.284

(d,

R-CO-N(-CH2-CH2-CH2-NH-CO-

CH(NHBoc)-CH2-CH2-CH2-CH2-NHBoc)2, 2H), 4.710 (s, R-CO-N(-CH2-CH2-CH2-NHCO-CH(NHBoc)-CH2-CH2-CH2-CH2-NHBoc)2, 2H), 4.157-4.139 (d, R-CO-N(-CH2-CH2CH2-NH-CO-CH(NHBoc)-CH2-CH2-CH2-CH2-NHBoc)2, 2H), 3.561-2.995 (m, R-CO-N(CH2-CH2-CH2-NH-CO-CH(NHBoc)-CH2-CH2-CH2-CH2-NHBoc)2, 12H), 2.275-2.237 (t, CH3-(CH2)10-CH2-CH2- of R group, 2H), 1.773-1.475 (m, CH3-(CH2)10-CH2-CH2-CO-N(CH2-CH2-CH2-NH-CO-CH(NHBoc)-CH2-CH2-CH2-CH2-NHBoc)2, 18H), 1.465 (bs, R-CON(-CH2-CH2-CH2-NH-CO-CH(NH-COO-C(CH3)3)-CH2-CH2-CH2-CH2-NH-COOC(CH3)3)2, 36H), 1.247 (bs, CH3-(CH2)10-CH2-CH2- of R group, 20H), 0.889-0.854 (t, CH3-(CH2)10-CH2-CH2- of R group, 3H); HR-MS (m/z): 998.7481 [(M+H)+] (Calculated), 998.7427 [(M+H)+] (Observed).

N,N-bis-[{3-(Boc-LLys-Boc)amido}propyl]hexadecanamide (2): Yield-66%;

1

H-NMR

(400 MHz, CDCl3) δ/ppm: 7.360-7.310 (d, R-CO-N(-CH2-CH2-CH2-NH-CO-CH(NHBoc)CH2-CH2-CH2-CH2-NHBoc)2, 2H), 5.437-5.333 (d, R-CO-N(-CH2-CH2-CH2-NH-COCH(NHBoc)-CH2-CH2-CH2-CH2-NHBoc)2, 2H), 4.751 (s, R-CO-N(-CH2-CH2-CH2-NHCO-CH(NHBoc)-CH2-CH2-CH2-CH2-NHBoc)2, 2H), 4.139-4.124 (d, R-CO-N(-CH2-CH2CH2-NH-CO-CH(NHBoc)-CH2-CH2-CH2-CH2-NHBoc)2, 2H), 3.526-2.985 (m, R-CO-N(CH2-CH2-CH2-NH-CO-CH(NHBoc)-CH2-CH2-CH2-CH2-NHBoc)2, 12H), 2.258-2.220 (t, CH3-(CH2)12-CH2-CH2- of R group, 2H), 1.715-1.459 (m, CH3-(CH2)12-CH2-CH2-CO-N(CH2-CH2-CH2-NH-CO-CH(NHBoc)-CH2-CH2-CH2-CH2-NHBoc)2, 18H), 1.404 (bs, R-CON(-CH2-CH2-CH2-NH-CO-CH(NH-COO-C(CH3)3)-CH2-CH2-CH2-CH2-NH-CO OC(CH3)3)2, 36H), 1.228 (bs, CH3-(CH2)12-CH2-CH2- of R group, 24H), 0.870-0.836 (t,



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CH3-(CH2)12-CH2-CH2- of R group, 3H); HRMS (m/z): 1026.7794 [(M+H)+] (Calculated), 1026.7795 [(M+H)+] (Observed).

N,N-bis-[{3-(Boc-DLys-Boc)amido}propyl]tetradecanamide (3): Yield-67%;

1

H-NMR

(400 MHz, CDCl3) δ/ppm: 7.311 (s, R-CO-N(-CH2-CH2-CH2-NH-CO-CH(NHBoc)-CH2CH2-CH2-CH2-NHBoc)2,

2H),

5.345-5.270

(d,

R-CO-N(-CH2-CH2-CH2-NH-CO-

CH(NHBoc)-CH2-CH2-CH2-CH2-NHBoc)2, 2H), 4.708 (s, R-CO-N(-CH2-CH2-CH2-NHCO-CH(NHBoc)-CH2-CH2-CH2-CH2-NHBoc)2, 2H), 4.159-4.142 (d, R-CO-N(-CH2-CH2CH2-NH-CO-CH(NHBoc)-CH2-CH2-CH2-CH2-NHBoc)2, 2H), 3.569-2.984 (m, R-CO-N(CH2-CH2-CH2-NH-CO-CH(NHBoc)-CH2-CH2-CH2-CH2-NHBoc)2, 12H), 2.277-2.238 (t, CH3-(CH2)10-CH2-CH2- of R group, 2H), 1.748-1.430 (m, CH3-(CH2)10-CH2-CH2-CO-N(CH2-CH2-CH2-NH-CO-CH(NHBoc)-CH2-CH2-CH2-CH2-NHBoc)2, 18H), 1.424 (bs, CH3(CH2)10-CH2-CH2-CO-N(-CH2-CH2-CH2-NH-CO-CH(NH-CO-OC(CH3)3)-CH2-CH2-CH2CH2-NH-COO-C(CH3)3)2, 36H), 1.247 (bs, CH3-(CH2)10-CH2-CH2- of R group, 20H), 0.889-0.855 (t, CH3-(CH2)10-CH2-CH2- of R group, 3H); HRMS (m/z): 998.7481 [(M+H)+] (calculated), 998.7413 [(M+H)+] (observed).

N,N-bis-[{3-(Boc-DLys-Boc)amido}propyl]hexadecanamide (4): Yield-78%;

1

H-NMR

(400 MHz, CDCl3) δ/ppm: 7.362-7.309 (d, R-CO-N(-CH2-CH2-CH2-NH-CO-CH(NHBoc)CH2-CH2-CH2-CH2-NHBoc)2, 2H), 5.435-5.345 (d, R-CO-N(-CH2-CH2-CH2-NH-COCH(NHBoc)-CH2-CH2-CH2-CH2-NHBoc)2, 2H), 4.753 (s, R-CO-N(-CH2-CH2-CH2-NHCO-CH(NHBoc)-CH2-CH2-CH2-CH2-NHBoc)2, 2H), 4.121 (s, R-CO-N(-CH2-CH2-CH2NH-CO-CH(NHBoc)-CH2-CH2-CH2-CH2-NHBoc)2, 2H), 3.529-2.979 (m, R-CO-N(-CH2CH2-CH2-NH-CO-CH(NHBoc)-CH2-CH2-CH2-CH2-NHBoc)2, 12H), 2.257-2.218 (t, CH3(CH2)12-CH2-CH2- of R group, 2H), 1.713-1.461 (m, CH3-(CH2)12-CH2-CH2-CO-N(-CH2CH2-CH2-NH-CO-CH(NHBoc)-CH2-CH2-CH2-CH2-NHBoc)2, 18H), 1.402 (bs, CH3(CH2)12-CH2-CH2-CO-N(-CH2-CH2-CH2-NH-CO-CH(NH-CO-OC(CH3)3)-CH2-CH2-CH2CH2-NH-COO-C(CH3)3)2, 36H), 1.226 (bs, CH3-(CH2)12-CH2-CH2- of R group, 24H), 0.868-0.834 (t, CH3-(CH2)12-CH2-CH2- of R group, 3H); HRMS (m/z): 1026.7794 [(M+H)+] (calculated), 1026.7742 [(M+H)+] (observed).

General procedure for synthesizing L-LANA-14, L-LANA-16, D-LANA-14 and DLANA-16: At first, 1-4 were dissolved in DCM at RT. To these solution then excess amount of trifluoroacetic acid (TFA) was added by mentioning 50% concentration in the reaction mixture. After that, the reaction was allowed to stir 2 h. At the end, the solvent from reaction


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mixture was removed by using rotory evaporator to accomplish the compounds with 100% yield. L-LANA-14: 1H-NMR (400 MHz, DMSO-d6) δ/ppm: 8.635-7.894 (m, R-CO-N(-CH2-CH2CH2-NH-CO-CH(NH3+)-CH2-CH2-CH2-CH2-NH3+)2, 14H), 3.715 (s, R-CO-N(-CH2-CH2CH2-NH-CO-CH(NH3+)-CH2-CH2-CH2-CH2-NH3+)2, 2H), 3.255-2.745 (m, R-CO-N(-CH2CH2-CH2-NH-CO-CH(NH3+)-CH2-CH2-CH2-CH2-NH3+)2,

12H),

2.254-2.220

(t,

CH3-

(CH2)10-CH2-CH2- of R group, 2H), 1.691-1.331 (m, CH3-(CH2)10-CH2-CH2-CO-N(-CH2CH2-CH2-NH-CO-CH(NH3+)-CH2-CH2-CH2-CH2-NH3+)2, 18H), 1.235 (bs, CH3-(CH2)10CH2-CH2- of R group, 20H), 0.867-0.833 (t, CH3-(CH2)10-CH2-CH2- of R group, 3H); HRMS (m/z): 598.53836 [(M+H)+] (calculated), 598.53872 [(M+H)+] (observed). L-LANA-16: 1H-NMR (400 MHz, DMSO-d6) δ/ppm: 8.618-7.855 (m, R-CO-N(-CH2-CH2CH2-NH-CO-CH(NH3+)-CH2-CH2-CH2-CH2-NH3+)2, 14H), 3.708 (s, R-CO-N(-CH2-CH2CH2-NH-CO-CH(NH3+)-CH2-CH2-CH2-CH2-NH3+)2, 2H), 3.257-2.762 (m, R-CO-N(-CH2CH2-CH2-NH-CO-CH(NH3+)-CH2-CH2-CH2-CH2-NH3+)2,

12H),

2.255-2.219

(t,

CH3-

(CH2)12-CH2-CH2- of R group, 2H), 1.709-1.327 (m, CH3-(CH2)12-CH2-CH2-CO-N(-CH2CH2-CH2-NH-CO-CH(NH3+)-CH2-CH2-CH2-CH2-NH3+)2, 18H), 1.235 (bs, CH3-(CH2)12CH2-CH2- of R group, 24H), 0.868-0.835 (t, CH3-(CH2)12-CH2-CH2- of R group, 3H); HRMS (m/z): 626.56966 [(M+H)+] (calculated), 626.57006 [(M+H)+] (observed). D-LANA-14: 1H-NMR (400 MHz, DMSO-d6) δ/ppm: 8.632-7.878 (m, R-CO-N(-CH2-CH2CH2-NH-CO-CH(NH3+)-CH2-CH2-CH2-CH2-NH3+)2, 14H), 3.714 (s, R-CO-N(-CH2-CH2CH2-NH-CO-CH(NH3+)-CH2-CH2-CH2-CH2-NH3+)2, 2H), 3.256-2.761 (m, R-CO-N(-CH2CH2-CH2-NH-CO-CH(NH3+)-CH2-CH2-CH2-CH2-NH3+)2,

12H),

2.254-2.220

(t,

CH3-

(CH2)10-CH2-CH2- of R group, 2H), 1.690-1.313 (m, CH3-(CH2)10-CH2-CH2-CO-N(-CH2CH2-CH2-NH-CO-CH(NH3+)-CH2-CH2-CH2-CH2-NH3+)2, 18H), 1.236 (bs, CH3-(CH2)10CH2-CH2- of R group, 20H), 0.869-0.835 (t, CH3-(CH2)10-CH2-CH2- of R group, 3H); HRMS (m/z): 598.53836 [(M+H)+] (calculated), 598.53868 [(M+H)+] (observed). D-LANA-16: 1H-NMR (400 MHz, DMSO-d6) δ/ppm: 8.630-7.876 (m, R-CO-N(-CH2-CH2CH2-NH-CO-CH(NH3+)-CH2-CH2-CH2-CH2-NH3+)2, 14H), 3.711 (s, R-CO-N(-CH2-CH2CH2-NH-CO-CH(NH3+)-CH2-CH2-CH2-CH2-NH3+)2, 2H), 3.255-2.761 (m, R-CO-N(-CH2CH2-CH2-NH-CO-CH(NH3+)-CH2-CH2-CH2-CH2-NH3+)2, 12H), 2.253-2.218 (t, CH3(CH2)12-CH2-CH2- of R group, 2H), 1.690-1.333 (m, CH3-(CH2)12-CH2-CH2-CO-N(-CH2CH2-CH2-NH-CO-CH(NH3+)-CH2-CH2-CH2-CH2-NH3+)2, 18H), 1.234 (bs, CH3-(CH2)12-



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CH2-CH2- of R group, 24H), 0.866-0.833 (t, CH3-(CH2)12-CH2-CH2- of R group, 3H); HRMS (m/z): 626.56966 [(M+H)+] (calculated), 626.57002 [(M+H)+] (observed). Antibacterial Assay against Growing Planktonic MRSA.44,47-49 Compounds were assayed in broth micro-dilution method by following published protocol with little modification. Detailed protocol can be found in the Supporting Information.

Cytotoxicity Assay.47 Toxicity against RAW cell lines was determined by following reported protocol with little modification. Details of experimental protocol can be found in the Supporting Information.

Time-kill Kinetics against Growing Planktonic MRSA.48 Bacterial culture obtained after single colony grown for 6 h were assayed for time kill kinetics by following our published protocol. Detailed experimental protocol is provided in the Supporting Information.

Antibacterial Activity against Stationary Phase MRSA.48 This experiment was performed by following our published protocol. A detail of experimental protocol is provided in the Supporting Information.

Biofilm Eradication Assay.44,48 Antibiofilm activity of LANAs and FA were compared by performing MBEC (minimum biofilm eradication concentration) assay. MBEC assay was performed by following our reported protocol with modification. Detailed of experimental protocol is provided in the Supporting Information.

Quantification

of

Biofilm

Disruption

and

Visualization

Through

Confocal

Microscopy.44,48 In order to quantify the biofilm disruption by D-LANA-14, established


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biofilms were prepared on glass surface. After compound treatment disrupted biofilms were quantified by staining with crystal-violet and visualization was made after staining with the dye Syto-9. Experiments were performed by following reported protocol. Details of experimental protocol can be found in the Supporting Information.

Membrane Depolarization of Both Growing Planktonic and Stationary Phase MRSA.44 Membrane depolarization assay was performed by using membrane potential sensitive dye, DISC3(5). Both actively growing and stationary phase of four different strains of MRSA were assessed by following our reported experimental precool. Details of experimental protocol are provided in the Supporting Information.

In-vivo Anti-MRSA Activity against Murine Model of Skin-infections.46 This experiment was performed with a little modification of our published protocol. Details are provided in Supporting Information. Acute Dermal Toxicity.50 The acute dermal toxicity of D-LANA -14 was determined by following our published protocol. A detail of experimental protocol is provided in Supporting Information. Resistance Development Studies.44,47,48 In order to investigate the long-lasting anti-MRSA activity of D-LANA-14, propensity of developing resistance in MRSA were assayed with two MRSA strains; MRSA-ATCC and MRSA-R3889. For comparing the efficacy of DLANA-14, control antibiotic FA was also included in these studies. Reported experimental protocol was followed and details are provided in the Supporting Information.



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ACKNOWLEDGEMENTS We thank Prof. C.N.R. Rao (JNCASR) for his constant support and encouragement. M.M.K thanks CSIR for research fellowship. We thank Dr. R. Ravikumar of National Institute of Mental Health and Neurosciences, (Bengaluru, India) for kindly providing us with the clinically isolated MRSA strains. We thank Mr. U. Adhikary for his comments on the manuscript.

SUPPORTING INFORMATION Details of experimental protocols, spectral data (NMR and HR-MS spectra) and figures for cytotoxicity assay, membrane depolarization assay against growing planktonic cells and acute dermal toxicity.

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