PEPTOID MIMIC VIA A TETHER

PRIOR APPLICATION INFORMATION

The instant application claims the benefit of US Provisional Patent Application 62/484,995, filed April 13, 2017 and entitled "AMPHIPHILIC TOBRAMYCIN-LYSINE CONJUGATES SENSITIZE MULTIDRUG RESISTANT GRAM-NEGATIVE BACTERIA TO RIFAMPICIN AND MINOCYCLINE", the contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The global health crisis caused by the resurgence of multidrug resistant bacteria strains, once believed to have been defeated, have called for an urgent need to rethink the principle of antibacterial drug discovery, and the judicious deployment of our current arsenal. ¹ An FDA incentive of accelerated development and review process for breakthrough therapies ² encourages the need to find alternative or better use of currently approved drugs instead of developing entirely new scaffolds. The Generating Antibiotics Incentives Now (GAIN) Acts of 2012 that seek to extend the exclusivity of new antibiotics ³ further stimulates the need to consolidate on the valuable knowledge of existing antibacterial scaffolds as a way out of the current attrition of drug candidates. Among the most recalcitrant bacteria, typified by the ESKAPE pathogens, ⁴

Pseudomonas aeruginosa, an opportunistic pathogen that commonly affects

immunocompromised patients, is particularly infamous for its highly sophisticated intrinsic and acquired resistance machineries. ⁵ ' ⁶ Compared to other bacteria, P.

aeruginosa displays low membrane permeability which limits the penetration of most antibiotics into the cell and a highly efficient membrane-associated efflux system of broad substrate specificity that significantly reduces bioaccumulation of drugs within its cytosol. ⁷ ' ⁸ The reduced intracellular concentration further promotes the activation of secondary adaptive resistance mechanisms (such as overexpression of efflux pump proteins and a variety of sensor kinases) that renders it completely refractory to treatment. ⁹ In addition, resistance often emerges when antibiotics are administered as monotherapy, ¹⁰ - ¹¹ thus combination therapy being the preferred choice in the treatment of complicated infections. ¹² ' ¹³ Although debatable, the argument for combination drug treatment is premised on the large-scale genetic interaction networks between targets. ¹⁴ The use of two or more antibiotics that impact multiple targets simultaneously, or adjuvants that aid the action of legacy antibiotics can indeed extend the antimicrobial space as well as mitigate the development of antibiotic resistance. ¹¹ Unfortunately, rational combination regimens and correlative synergistic mechanisms have remained largely unexplored and clinical benefits are yet to be demonstrated. Since the outer membrane of P. aeruginosa remains a major impediment to the influx of antibiotics, we and others have been investigating the effects of adjuvants that perturb this

'impermeable' layer, and consequently synergize the activities of other antibiotics. For instance, Evotec AG and Spero Therapeutics are currently developing a polymyxin- based antimicrobial peptide SPR-741 as a potentiator to overcome outer membrane impermeability of legacy antibiotics against Gram-negative pathogens in clinical trials ¹⁵ . Our group has recently shown that tobramycin-fluoroquinolone hybrids interact with both the outer- and inner-membranes of P. aeruginosa resulting in enhanced cell penetration and reduced efflux by dissipating the proton motive force (PMF) that drive efflux pumps in P. aeruginosa. ⁶ ' ¹⁷ Mode of action studies indicate that the function of the tobramycin moiety in these hybrids is limited to a membrane-destabilizing effect of the outer membrane that results in self-promoted uptake of the hybrid and/or the antibiotic. In contrast, the function of the fluoroquinolone moiety in the hybrid is less clear but the adjuvant- and antibacterial properties appear to be correlated to the hydrophobic nature and membrane destabilizing effect of the fluoroquinolone group. ¹⁶ ' ¹⁷ Amphiphilic aminoglycosides (AAGs) that combine aminoglycosides such as tobramycin with alkyl or other hydrophobic groups have also been previously reported to show improved activity and different modes of action in killing pathogens, compared to their constituent parent antibiotics. ¹⁸ For example, some amphiphilic tobramycin derivatives were demonstrated to primarily target the bacterial membrane, ^19-21 as well as possess immumodulatory properties that closely resemble that of the natural host defense peptides. ²² SUMMARY OF THE INVENTION

According to an aspect of the invention, there is provided a compound comprising tobramycin connected by a tether to a lysine-based peptoid mimic.

According to another aspect of the invention, there is provided a method of treating a bacterial infection in an individual in need of such treatment comprising administering to said individual an effective amount of a compound comprising tobramycin connected by a tether to a lysine-based peptoid mimic.

According to another aspect of the invention, there is provided a method of preparing a medicament for treatment of a bacterial infection comprising admixing a compound comprising tobramycin connected by a tether to a lysine-based peptoid mimic and a suitable excipient.

According to a further aspect of the invention, there is provided a compound comprising tobramycin connected by a tether to a lysine-based peptoid mimic for treating a bacterial infection.

According to another aspect of the invention, there is provided use of a compound comprising tobramycin connected by a tether to a lysine-based peptoid mimic for treating a bacterial infection.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Structures of tobramycin, reference peptoid (4), and amphiphilic tobramycin-lysine conjugates with varied alkyl tether (1-3).

Figure 2. Absolute MIC of minocycline or rifampicin alone or in combination with 4 pg/mL of compound 1- 4 against P. aeruginosa PAO1.

Figure 3. FIC index comparison of compound 3 and colistin in combination with antibiotics against P. aeruginosa PAO1 .

Figure 4. Time killing kinetics of minocycline (MIN) or rifampicin (RMP) alone at 1/2 x MIC (4 pg/mL for MIN and RMP for 8 pg/mL) or in combination with compound 3 against P. aeruginosa PAO1 at varied concentrations.

Figure 5. Emergence of bacterial resistance after treatment of P. aeruginosa PAO1 with antimicrobials for 25 passages at sub-MIC concentration was determined in monotherapy of tobramycin, colistin, minocycline (MIN), and rifampicin (RMP) or in combination therapy of MIN plus compound 3 or RMP plus compound 3.

Figure 6. Outer membrane permeabilization of P. aeruginosa PAO1 by colistin, compound 3, or 4 at 32 pg/mL was determined using fluorescence dye CFDA at an excitation wavelength of 488 nm and an emission wavelength of 520 nm. 1 % Triton X- 100 was served as positive control.

Figure 7. Cytoplasmic membrane depolarization of P. aeruginosa PAO1 treated with tobramycin, colistin, compound 3, or 4 at 32 pg/rriL was measured using the membrane potential-sensitive dye diSC3-5. The fluorescent intensity was monitored at an excitation wavelength of 622 nm and an emission wavelength of 670 nm over a 1200 seconds period.

Figure 8. Swimming motility of P. aeruginosa PAO1 treated with compound 3 or 4 at 4 pg/mL.

Figure 9. (A) Hemolytic activity of compounds 1-4 was evaluated against pig red blood cells. 0.1 % Triton X-100 was employed as positive control to calculate the percentage of hemolysis. (B) Cytotoxicity of compound 3 was demonstrated against DU145 and JIMT-1 cell lines by MTS assay.

Figure 10. Evaluation of monotherapy and combination therapy in protecting Galleria mellonella larvae from XDR P. aeruginosa #P262 infection. MIN = minocycline; RMP = rifampicin.

Figure 1 1 . Scheme 1 . General synthetic scheme for the preparation of amphiphilic peptoid.

Figure 12. Scheme 2. General synthetic scheme for the preparation of amphiphilic tobramycin-lysine conjugates.

Figure 13 - Time killing kinetics of minocycline, rifampicin, and 3 as monotherapy against P. aeruginosa PAO1 at 1 MIC, 2 χ MIC, and 4 MIC, respectively.

Figure 14 - Tolerability of Galleria mellonella treated with compound 3 and 4 at 100 and 200 mg/kg. The numbers of surviving larvae were scored daily for 4 days.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.

To preserve the amphiphilic nature of the membrane-active tobramycin derivatives, it is imperative to carefully evaluate the hydrophobic nature of the moiety to be attached. Recently, Haldar and co-workers described a series of ultrashort antibacterial lysine-based peptoid mimics that contain facial segregation of positively- charged L-lysine, hydrophobic aromatic core, and alkyl chain. ²³ These molecules were reported to facilitate the attraction of compounds to bacterial surfaces and permeate cell membrane, and displayed some promising antimicrobial properties. ²³ We reasoned that the inherent amphipathic nature of these peptoids, combined with their antibacterial properties could be amplified by linking to a tobramycin-based vector as previously reported for tobramycin-fluoroquinolone hybrid antibiotics. ¹⁶ ' ¹⁷

We designed a series of tobramycin analogs 1-3 by conjoining an amphiphilic peptoid mimic 4 to the C-5 position of tobramycin, with varied alkyl tether, to investigate the adjuvant property of the resultant conjugates in combination with commonly used antibiotics (Figure 1 ). The synergistic effects of the tobramycin-lysine conjugates in combination with various classes of antibiotics, against P. aeruginosa (wild-type and clinical isolates) were determined using checkerboard study. The emergence of bacterial resistance was compared between single agent and combination therapy for 25 bacterial passages, and Galleria mellonella infection worm model was further used to assess in vivo synergistic benefits of the optimal drug combination that protects P. aerug/nosa-challenged larvae. Hemolysis and cytotoxicity assays were carried out to ascertain toxicity against mammalian cells, and prokaryotic membrane-compound interactions were studied to gain mechanistic insights on possible modes of action.

As discussed herein, we demonstrated that amphiphilic tobramycin-lysine conjugates preserve many of the known adjuvant properties of previously reported tobramycin-fluoroquinolone hybrids. From a medicinal chemistry point, linking a tobramycin C-12 vector to an amphiphilic lysine conjugate enhances the outer membrane destabilization effect of the amphiphilic lysine analog. As such our study suggests that a tobramycin-C12 tether at C-5 position in tobramycin serves as an effective vector to promote delivery of compounds through the outer membrane barrier of Gram-negative bacteria with an optimized effect on P. aeruginosa. However, the effect of the tobramycin-C12 tether appears to be not limited to the outer membrane but also involves the cytoplasmic membrane. For instance, we provide evidence that conjugates containing a tobramycin-C12 tether reduces the ΔΨ component of the PMF located at the cytoplasmic membrane. This leads to decreased activity of the efflux associated pumps but at the same time can lead to enhanced cytoplasmic uptake of agents which depend on the ΔρΗ component of the PMF like the tetracycline class of antibiotics. Overall, this study provides a promising strategy for generating effective antibiotic adjuvants that overcome drug resistance in MDR Gram-negative bacteria including P. aeruginosa by carefully designing amphiphilic tobramycin tethers. The discovery that compound 3 can potentiate several classes of antibiotics against resistant pathogens is set to expand the antimicrobial space and optimize our usage of antibiotics in our current armamentarium.

Bacterial resistance can frequently emerge in antibiotic monotherapy due to the selective pressure that naturally separates out the resistant phenotypes. ^{10 11}

Combination of two or more antimicrobials that can impact multiple targets simultaneously is believed to be capable of suppressing drug resistance, as well as broaden the spectrum of activity of a treatment course than single agents. ^32,33 In the last two years the FDA has approved two new combination drugs Avycaz (ceftazidime + avibactam) and Zerbaxa (ceftolozane + tazobactam to combat MDR gram-negative infections). Ceftazidime/avibactam contains an older 3 ^rd generation cephalosporin ceftazidime, with avibactam a synthetic ηοη-β-lactam, β-lactamase inhibitor that inhibits the activities of Ambler class A and C β-lactamases and some Ambler Class D enzymes. ³⁴ Limited data suggest that the addition of avibactam does not improve the activity of ceftazidime versus Pseudomonas aeruginosa. Ceftolozane is a novel cephalosporin with a chemical structure similar to that of ceftazidime, with the exception of a modified side-chain at the 3-position of the cephem nucleus, which confers potent antipseudomonal activity. ³⁵ The addition of tazobactam extends the activity of ceftolozane to include most ESBL producers but not P. aeruginosa. Nevertheless, effective drug combinations often lead to inconclusive benefits of combination therapy over monotherapy during meta-analysis. ³⁶ Recent reports about the potentials of amphiphilic tobramycin analogues to permeabilize cell membrane, ¹⁹ ' ²⁰ and our previous studies that demonstrated the intrinsic ability of tobramycin-fluoroquinolone hybrids to potentiate the antimicrobial activity of several classes of antibiotics against clinical P. aeruginosa isolates, ¹⁶ - ¹⁷ encouraged a further optimization of this promising scaffold for use as adjuvants.

As discussed herein, we prepared new amphiphilic tobramycin hybrids by taking advantage of the membrane-active peptoid 4 as a modulator. The antimicrobial properties of these derivatives alone were assessed and demonstrated to be weaker compared to the parental tobramycin molecule (Table 1 ). Although tobramycin is believed to induce pleiotropic mechanisms of action, ³⁷ the most acceptable hypothesis suggests tobramycin permeates the outer membrane via a self-promoted uptake mechanism ³⁸ and acts by impairing bacterial protein synthesis through irreversible binding to the 30S ribosomal subunit. ³⁹ The differing activity between tobramycin and the newly synthesized hybrid molecules suggests that the protein translation inhibitory effect is compromised by attachment of hydrophobic moieties to tobramycin as previously shown. ¹⁶ ' ⁷ Furthermore, activity trend between the hybrids revealed a correlation between antimicrobial potency and carbon chain length. In general, the longer the carbon tether, the better the antimicrobial efficacy of the compound. As discussed herein, in some embodiments, the tether is a tether that has a length corresponding to 2-20 carbons. As will be appreciated by one of skill in the art, such a tether could be not only alkyl based but could be composed of anything that can approximate this length such as for example but by no means limited to aromatic tethers, polyethyleneglycol based or carbon chains with double or triple bonds. The physicochemical properties necessary to navigate a complex membrane topology, especially as represented in P. aeruginosa, is perhaps the principal reason for the varied activity of the hybrid molecules. Although ultrashort and amphiphilic lysine-based peptoid mimics were previously reported to have promising activities against P. aeruginosa MTCC 424, ²³ our evaluation of compound 4 (different alkyl chain) against a panel of organisms revealed otherwise (Table 1 ). This may be due to the slight change in the length of the alkyl chain (C10 to C12), and perhaps, the different bacterial strains tested. The amphiphilic nature of 4, the reported properties of tobramycin hybrids, ¹⁶ and the differential activity of the new molecules based on carbon chain length, gave a clue on possible membrane effect of these compounds. Thus, we investigated antimicrobial activities of the amphiphilic conjugates in combination with other antibiotics, particularly against P. aeruginosa, the major nosocomial pathogen and leading cause of infection in cystic fibrosis patients. Although most of the clinical isolates investigated in this study were resistant to tobramycin and other antibiotics (with the exception of colistin), the MIC values of the conjugates against these strains were similar to that of wild-type P. aeruginosa PA01 (Table 2), suggesting that the targeting site and/or mechanism of action of the conjugates was different than that of tobramycin. For instance, amphiphilic tobramycin-lysine conjugate 4 possesses similar MICs against the eight strains (range 8-64 μg/mL) while tobramycin possesses a MIC range between 0.25 - 512 g/mL indicating that the conjugate is 32-fold less active against tobramycin susceptible strains while being 16-fold less active against tobramycin-resistant strains which supports a different mode of action.

The ability of 3 to perturb the membrane was verified by its synergistic effect with vancomycin, a drug that cannot pass through the outer membrane of P. aeruginosa due to its large size (M.wt. = 1449.2). Synergism was also observed for other antibiotics with different modes of action against P. aeruginosa, the most prominent being novobiocin, minocycline and rifampicin. Importantly, combining 3 with minocycline, or with rifampicin, can revive the antimicrobial activities of these antibiotics against MDR and XDR P. aeruginosa isolates (Table 4). The uptake of tetracyclines, such as minocycline, is known to be driven by transmembrane chemical gradient (ΔρΗ) of PMF generated by the respiratory chain on the cytoplasmic membrane. ⁴² The other component of PMF is electrical potential (ΔΨ), which is known to drive aminoglycosides uptake. ⁴³ Bacteria control ΔΨ and ΔρΗ exquisitely to maintain a constant value of PMF, and disruption of either component is compensated for by a counteracting increase in the other. ⁴⁴ When a compound disrupts ΔΨ, an antagonism effect will be observed in combination with aminoglycosides, while synergism will show in combination with tetracyclines due to the compensatory increase of ΔρΗ. Tetracyclines and aminoglycosides have therefore been used as two relevant antibiotics in combination studies with other drugs to identify compounds that affect membrane PMF and specifically dissipate either component of PMF. ³¹ In this study, compound 3 displayed different synergistic effects with minocycline (synergism) and tobramycin (no interaction), an observation that is consistent with dissipation of ΔΨ component of the PMF by 3. However, the expected antagonistic effect of 3 with aminoglycosides was not observed, likely due to the membrane penetration induced by 3 that slightly affected aminoglycosides uptake into bacterial cells. The effect of 3 on ΔΨ was further corroborated by the increased diSC3-5 fluorescence (Figure 7) and repression of swimming motility controlled by this parameter (Figure 8). Compounds that collapse the PMF are known to inhibit ATP synthesis and flagellar motility, preventing or reducing swimming activity. ⁴⁵

Membrane-associated efflux is another major mechanism that prevents bioaccumulation of drugs within the cytosol, thus preventing/reducing access of antibiotics to intracellular targets. ⁴⁶ Efflux pump proteins localized in the cytoplasmic and outer membrane, and linked by a periplasmic membrane fusion protein (MFP), play a major role in intrinsic and acquired resistance of P. aeruginosa. ^7'9 The associated resistance is based on energy-dependent effluxes, which are usually driven by PMF. ³⁰ We envisaged that the dissipation of ΔΨ in PMF will prevent electron transport across the respiratory chain, thus inhibiting ATP synthesis and ultimately affect efflux pump system. We therefore studied the effect of the conjugates on efflux pumps using P. aeruginosa efflux-deficient strains PAO200 and PAO750, and perhaps explain the observed synergistic mechanism more exquisitely. The efflux pump knock-out decreased the MIC of minocycline by 8-fold (Table 5), confirming that minocycline is a substrate for these efflux pumps, which is consistent with previous study ⁷ The synergism observed when 3 was combined with minocycline against wild-type PA01 was not observed in PAO200 and PAO750, with FIC indices > 0.5, indicating that the antimicrobial activity potentiation of minocycline by 3 is due to the inhibition of efflux pumps, particularly the RND pumps. To validate this hypothesis, the synergistic effects of 3 with other known P. aeruginosa efflux pump substrates were evaluated in PAO200 and PAO750, including chloramphenicol, erythromycin, trimethoprim, and moxifloxacin. ⁷ ' ^48-50 The results showed weak synergy or additive effects of 3 in these combinations, corroborating the efflux pump inhibitory activity of 3. We posited that the dissipation of electrochemical gradient across the cytoplasmic membrane affected respiratory ATP production, thereby compromising efflux pump efficiency.

Surprisingly, rifampicin, which is not substrate of the five efflux pumps investigated in this study (MexAB-OprM, exCD-OprJ, MexEF-OprN, MexJK, and MexXY) was similarly strongly synergized by 3 against PAO200 and PAO750 (Table 5). Rifampicin is known to kill bacteria by inhibiting RNA synthesis after binding to DNA- dependent RNA polymerase. ⁵¹ Both gram-positive and gram-negative bacteria are similarly sensitive to rifampicin, with the higher MICs reported in gram-negative bacteria due to its low penetration across the outer membrane. ⁵² A combination study of rifampicin with colistin (a well-known membrane permeabilizer) has demonstrated that perturbation of P. aeruginosa outer membrane can indeed potentate the antimicrobial activity of rifampicin, ⁵³ thus confirming the results of this study with 3 (Figure 3). Outer membrane perturbation is perhaps the reason why 3 was able to synergize rifampicin against PAO200 and PAO750 despite not being a substrate for the pumps. It is however clear that minocycline is more sensitive to PMF dissipation caused by compound 3 than simply membrane penetration induced by colistin, as evident in the FIC indices > 0.5 shown in Table 5. These results suggest that compound 3 is not just able to penetrate P. aeruginosa cell membrane like colistin, but could also dissipate the cytoplasmic membrane, and compromise the efficient functioning of the efflux systems. Although reference compound 4 similarly displayed cytoplasmic membrane depolarization activity and suppressed bacterial swimming motility, it was to a lesser extent than 3 at the same concentration (Figures 7 and 8), and did not display any synergistic effect in combination with minocycline and with rifampicin (Table 7). The inability of 4 to potentiate minocycline like 3 despite its ability to partially depolarize the cytoplasmic membrane as well may be due to its weak outer membrane perturbation that prevents uptake of other antimicrobial agents. The simultaneous occurrence of both phenomenon is indeed critical to the adjuvant properties of tobramycin-lysine conjugates.

In contrast to the bactericidal nature of rifampicin, minocycline is known to be only bacteriostatic, which was evident in the time-kill assay with constant bacterial cells number at all concentrations tested (Figure 13), the observation that is consistent with previous studies. ⁵⁴ ' ⁵⁵ However, the increased killing efficiency of minocycline when used in combination with 3 is likely attributable to the effect of 3 on the membrane. Attempts to select for resistance with combination of 3 and minocycline during 25 serial passages resulted in a 4-fold increase in MIC, as opposed to minocycline and tobramycin alone that had 16- and 256-folds increase respectively (Figure 5). Indeed, it is more difficult for bacteria to develop resistance to simultaneously-acting drug combination, especially when one of the drugs acts on the membrane ^56"58 .

A major concern about membrane-acting and PMF-collapsing agents is their toxicities towards eukaryotic cells. ³¹ To verify the safety of these compounds, the toxicities of the conjugates were evaluated against pig erythrocytes and mammalian cancer cell lines. It was surprising to see a dramatic reduction of the hemolytic activity of 4 when joined to tobramycin (Figure 9A). This is probably caused by changes to the molecular amphipathy as previously seen for antimicrobial peptides. ⁵⁹ Moreover, combination therapy would allow for reduced doses to be used, minimizing cytotoxicity, and 3 displayed negligible toxicity at its effective synergistic concentration (< 4 pg/mL). In the in vivo study, the high tolerance of Galleria mellonella worms to 3 (100% survival at 200 mg/kg after 96 h) further confirmed the safety of this compound. Galleria mellonella injection model has been commonly used in accessing the in vivo efficacy of antimicrobials against P. aeruginosa because it shares a high degree of structural and functional homology to the immune systems of vertebrates with both cellular and humoral defenses. ⁶⁰ In contrast to monotherapy, single dose combination of 3 (75 mg/kg) plus minocycline (75 mg/kg) or 3 (75 mg/kg) plus rifampicin (75 mg/kg) effectively protected larvae from XDR P. aeruginosa P262 infection with more than 75 % survival after 24 h, indicating the therapeutic potential of amphiphilic tobramycin as an adjuvant to treat infection caused by XDR P. aeruginosa.

Specifically, in one aspect of the invention, there is provided a compound comprising tobramycin connected by a tether to a lysine-based peptoid mimic. As will be appreciated by one of skill in the art, the lysine-based peptoid mimic may be an L-lysine or a D-lysine

In some embodiments, the lysine-based mimic comprises a positively-charged lysine, a hydrophobic aromatic core, and an alkyl chain. The lysine may be L-lysine D-lysine.

In some embodiments, the compound comprises a structure as set forth Formula (I):

(I).

wherein the lysine-based peptoid mimic is connected to the C-5 position of tobramycin by a tether where "n" is an integer between 2-20, indicating that the tether is of a length of between 2-20 carbons. As will be appreciated by one of skill in the art, this does not mean that the tether or linker necessarily needs to include 2-20 carbon molecules, merely that the tether or linker must be of the same approximate length as 2- 20 carbon molecules. As such, the tether may be for example but by no means limited to an alkyl-based tether, an aromatic-based tether, a polyethyleneglycol-based tether or carbon chains with for example double or triple bonds.

For example, the tether may comprise a length corresponding to 2-20, 3-20, 4- 20, 5-20, 6-20, 2-19, 2-18, 2-17, 2-16, 2-15, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 4-20, 4- 19, 4-18, 4-17, 4-16, 4-15, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 6-20, 6-19, 6-18, 6-17, 6- 15, 6-15, 6-14, 6-13, 6-12, 6-1 1 , 7-1 1 or 8-1 1 carbon atoms.

As will be appreciated by one of skill in the art, any suitable tether or linker known in the art may be used to connect the lysine-based peptoid mimic to the tobramycin provided that the tether is sufficiently flexible and sufficiently long that the tobramycin and the lysine-based peptoid mimic are able to function as discussed herein.

In some embodiments, the tether is a flexible tether, as shown in Figure 1 . For example, the tether may comprise 3-11 repeating alkyl units.

In some embodimen the compound may be one of compounds 1-3:

According to another aspect of the invention, there is provided a method of treating a bacterial infection in an individual in need of such treatment comprising administering to said individual an effective amount of a compound comprising tobramycin connected by a tether to a lysine-based peptoid mimic. As will be appreciated by one of skill in the art, the symptoms associated with a bacterial infection are well-known to those of skill in the art. As such, a "person in need of such treatment" is a person who has or is suspected of having a bacterial infection, particularly a bacterial infection that has been difficult to treat with conventional or prior art antibiotics alone or is a bacterial infection that is suspected of or known to be caused by a bacterial strain that may be drug resistant.

In some embodiments, the bacterial infection is caused by a pathogenic gram- negative bacteria. The pathogenic gram-negative bacteria may be for example but by no means limited to E. coli, Acetinobacter, Salmonella, Shigella, Enterobacteriaceae, Pseudomonas, Moraxella, Heliobacter, Stenotrophomonas, Bdellovibrio, Legionella and the like. In some embodiments, the pathogenic gram-negative bacteria is P. aeruginosa, E. coli or Acetinobacter baumannii. In some embodiments, the P. aeruginosa strain is an extremely drug-resistant strain of P. aeruginosa.

As will be appreciated by one of skill in the art, an "effective amount" is an amount that is sufficient to accomplish at least one of the following: reduction in the severity of the symptoms associated with the bacterial infection; and reduction in the CFU per ml of bacteria in a sample taken from the individual. As discussed herein, this may be accomplished by increasing or enhancing the anti-bacterial activity of another compound. For example, while not wishing to be bound to a particular theory or hypothesis, the inventors believe that the compounds described herein enhance the permeability of the gram-negative outer membrane and/or inner membrane and thereby in effect act as a delivery system. As such, in these embodiments, an "effective amount" is an amount that is sufficient to enhance the permeability of the outer membrane of the infective bacteria so that an antibacterial agent can enter the bacterial cell. As will be appreciated by one of skill in the art, this means that a wide variety of compounds that have bacteria targets but cannot cross the outer membrane on their own can now be considered to be anti-bacterial agents when used in combination with the compounds of the invention. Furthermore, other anti-bacterial agents may be administered at a much lower effective amount, as discussed herein.

Other methods for determining an effective amount of the compound to be administered to the individual will be apparent to one of skill in the art and may depend on many other factors, for example, the age and general condition of the individual and the severity of the infection and/or symptoms associated with the infection.

As discussed herein, the compound may be co-administered with an antibacterial compound such as an antibiotic. As will be appreciated by one of skill in the art, the compound and the antibiotic may be co-administered simultaneously although this is not necessary.

In another embodiment of the invention, there is provided use of a compound comprising tobramycin connected by a tether to a lysine-based peptoid mimic as discussed above for enhancing the antibacterial activity of an agent. As discussed herein, it is important to note that the agent does not necessarily have antibacterial properties on its own and may need to be co-administered with the compound in order to enter the bacterial cell. Alternatively, the agent may have antibacterial properties on its own but these antibacterial properties may be enhanced by increased entry of the agent into the bacterial cell by the membrane permeability enhancing activity of the agent, as discussed herein.

According to another aspect of the invention, there is provided a method of preparing a medicament for treatment of a bacterial infection comprising admixing a compound comprising tobramycin connected by a tether to a lysine-based peptoid mimic and a suitable excipient such as for example but by no means limited to a pharmaceutically acceptable excipient, which are well-known in the art. The compound may be formulated for co-administration with an agent having anti-bacterial properties either alone or when administered in combination with the compound, as discussed herein. For example, the compound and the antibiotic of choice may be formulated as a single unit dosage or the compound and the antibiotic of choice may be separate unit doses but may be otherwise packaged together.

For example, in some embodiments, the antibiotic of choice, that is, the coadministered antibiotic may be a tetracycline.

Alternatively, the antibiotic may be an antibiotic against which bacterial resistance is mediated by efflux pump activity.

In some embodiments, the antibiotic is selected from the group consisting of fluoroquinolones (such as for example ciprofloxacin, moxifloxacin, and levofloxacin), tetracyclines (such as for example minocycline, tetracycline, tigecycline, and oxycycline), fosfomycin, anthelminthic drugs (such as for example benzimidazoles (such as for example albendazole, thiabendazole), niclosamide, and oxyclozanide), rifampicin, macrolides (such as for example erythromycin and others), sulfadrugs (such as for example sulfamethoxazole), trimethoprim, vancomycin and combinations thereof.

In some embodiments, the antibiotic is selected from the group consisting of: moxifloxacin; novobiocin; minocycline; rifampicin; chloramphenicol; erythromycin; trimethoprim; and vancomycin.

The invention will now be further explained and elucidated by way of examples; however, the invention is not necessarily limited by the examples.

EXAMPLE 1 - Synthesis

The reference compound ultrashort peptoid mimic 4 was synthesized by reductive amination of aromatic aldehyde 5 with dodecylamine 6 generating secondary amine 7 which after coupling to di-Boc-protected lysine produced protected lysine- based peptoid 8. Deprotection of the Boc-protecting groups with TFA afforded ultrashort lysine peptoid 4 as previously described (Figure 1 1 ). ²³ The synthesis of tobramycin- lysine conjugates 1-3 was achieved by preparing amphiphilic tobramycin derivatives (5 steps), followed by a single-step reductive amination conjoining and a final deprotection (Figure 12). Preparation of the tobramycin derivatives commenced by protecting the amines on commercially available tobramycin with Boc anhydride, followed by silylation of the hydroxyl groups with a bulky protecting group such as TBDMSCI to give 9. This is to ensure the more hindered C-5 position of the Boc- and TBDMS-protected tobramycin intermediate, the desired point of alkylation, is unprotected. This is the preferred position because C-5-modified tobramycin derivatives retained antibacterial activity ²⁴ and superior adjuvant properties against gram-negative bacteria like P. aeruginosa. ^{' 6} - ¹⁷ The C-5 hydroxyl group of 9 was subsequently alkylated with 1 ,π-dibromoalkane (n = 4, 8, 12), under phase-transfer catalytic conditions, to afford alkylated tobramycin analogs 10a-c. The terminal bromo-group of intermediate 10a-c was then displaced by an azido nucleophile under anhydrous conditions to give 11a-c, followed by reduction to free amine 12a-c via catalytic hydrogenation. This free amine was successively reacted with commercially-available 10-chloro-9-anthracenaldehyde via reductive amination, to afford intermediate 13a-c with secondary amine, which was then coupled to di-Boc- protected Lys to produce protected amphiphilic tobramycin amides 14a-c. The final deprotection step involved the removal of Boc and TBDMS protecting groups using MeOH:HCI (3:2, v/v) to afford the desired target compounds 1 - 3. The final compounds were characterized by NMR, mass spectrometry, and reverse-phase HPLC, with > 95 % purity.

EXAMPLE 2 - Antimicrobial activity

The antimicrobial activities of reference peptoid mimic (compound 4) and tobramycin hybrids 1-3 against a panel of gram-negative and gram-positive bacteria are presented as the minimum inhibitory concentration (MIC) in Table 1. Reference peptoid mimic 4 with a C12 hydrophobe displayed weaker antimicrobial activity compared to the reported C10 peptoid, ²³ with MIC > 8 pg/mL against all the strains tested in this study. For tobramycin-lysine conjugates, there was a positive correlation between antimicrobial activity and the length of the carbon chain tether. Compound 3 with C12 tether was the most potent analog of all the hybrids, and displays moderate activity against gram- positive bacteria (MIC of 2-32 pg/mL) but a relatively weak activity against gram- negative bacteria (MIC > 16 pg/mL). Further, the anti-pseudomonal activities of all compounds were evaluated against wild-type and seven clinical isolates of P. aeruginosa, including MDR, XDR, and colistin-resistant strains (Table 2). A similar trend of longer carbon chain displaying better activity was observed for drug-resistant P. aeruginosa as well, suggesting C12 as the optimal tether length of all analogues tested. In addition, we observed comparable activity of 3 against wild-type and drug-resistant strains (MIC ranging from 8 to 64 pg/mL) indicating that compound 3 is not greatly affected by resistance. Again, compound 4 did not show potent activity against any of the clinical isolates tested, with MIC > 64 pg/mL. The MIC of tobramycin increased from 0.25 pg/mL in wild-type P. aeruginosa to > 8 pg/mL in drug-resistant strains, particularly in P262 (MIC = 512 pg/mL). Similar results were also observed for other tested antibiotics, with the exception of colistin. It is however interesting to note that compound 3 displayed better antibacterial activity against certain MDR P. aeruginosa strains when compared to the antipseudomonal agents ciprofloxacin and ceftazidime but also nonpseudomonal drugs such as moxifloxacin, minocycline, rifampicin, chloramphenicol, erythromycin, and trimethoprim (Table 2).

EXAMPLE 3 - Combination study of hybrids with antibiotics

To assess the adjuvant properties of the hybrids, checkerboard studies were performed to determine the synergistic effects of the most active hybrid 3 with 14 different antibiotics (cutting across all classes) against wild type P. aeruginosa PA01 . The fractional inhibitory concentration (FIC) index, a numerical quantification of the interactions between antibiotics, was calculated as previously described. ¹⁶ FIC indices of < 0.5, 0.5 - 4, and > 4 indicate synergy, no interaction, and antagonism respectively. ²⁵ Compound 3 showed strong synergy with most antibiotics tested against PA01 except ceftazidime, colistin, meropenem and the aminoglycosides gentamicin, kanamycin A and amikacin. The strongest potentiation was seen with novobiocin (FIC index = 0.071 ), minocycline and rifampicin (FIC index = 0.094 for both) as shown in Table 3. Synergism with minocycline and rifampicin was also observed with 2, but not with 1 , 4, and tobramycin (Table 7). As shown in Figure 2, the absolute MICs (the MIC of antibiotics in the presence of adjuvants at 4 pg/mL) of minocycline or rifampicin in combination therapy with hybrids was dramatically lower than monotherapy, in particular with compound 3 where MICs of minocycline and rifampicin were reduced from 8 pg/rnL and 16 pg/mL in monotherapy to 0.25 pg/rnL (32-fold potentiation) and 0.0625 pg/rnL (256- fold potentiation), respectively. Thus, combinations of 3 with minocycline and rifampicin were selected for further synergy studies against a panel of P. aeruginosa clinical isolates (Table 4). Compound 3 demonstrated strong synergy with minocycline and rifampicin across the clinical isolates panel (FIC indices of 0.039 to 0.281 ), with the exception of rifampicin against P. aeruginosa 91433 (FIC index = 0.5). Since the breakpoints for minocycline and rifampicin against P. aeruginosa are not available (as they are not conventional drugs for treating P. aeruginosa infections), the susceptible or intermediate resistant breakpoints of minocycline against Acinetobacter spp. and that of rifampicin against Enterococcus spp. reported by CLSI, ²⁶ were considered as interpretive MIC standards for this study. The susceptible breakpoints of minocycline (MIC < 4 pg/rnL) against Acinetobacter spp. were reached for all minocycline-resistant, MDR, or XDR P. aeruginosa isolates at 4 g/mL of 3. For rifampicin, susceptible (MIC < 1 Mg/mL) or intermediate resistant (MIC = 2 pg/rnL) breakpoints against Enterococcus spp. were reached in 5/7 rifampicin-resistant, MDR, or XDR P. aeruginosa isolates, with the exception of strain P262 and #91433. These results suggest that conjugate 3 can rescue minocycline in all tested isolates and rifampicin against most of the tested P. aeruginosa isolates from resistance.

To gain insights into the membrane effects and relevant synergistic mechanism of 3, colistin, a membrane-active antibiotic, was tested in combination with five antibiotics against PA01 . Our results indicated that colistin was also able to potentiate the activity of rifampicin and novobiocin (Figure 3), but to a lesser extent than compound 3. To investigate the relevance of efflux pumps on the observed adjuvant properties of 3, combination studies of minocycline and rifampicin each with compound 3 were carried out in efflux pump-mutated strains, PAO200 and PAO750. PAO200 is a MexAB-OprM deletion strain while PAO750 is an efflux-sensitive strain that lacks five different clinically-relevant RND pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexJK, and MexXY) and the outer membrane protein OpmH. Some of these pumps are homologues of broad substrate specificities that expel different classes of antimicrobial agents and confer resistance on P. aeruginosa. For instance, the tripartite protein system MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM allow the translocation of a wide variety of substrates such as quinolones, chloramphenicol, trimethoprim, imipenem, and tetracyclines out of the cell. ²⁷ As shown in Table 5, synergism was observed with rifampicin in both PAO200 (FIC index = 0.129) and PAO750 (FIC index = 0.156), but not with minocycline (FIC index > 0.5). The ability to potentiate minocycline in PAO1 but not in efflux-deficient strains corroborates the hypothesis that tobramycin-lysine conjugate compromises the efficiency of efflux proteins. Rifampicin, which is not a substrate for these pumps was however potentiated, suggesting a second mode of action consistent with membrane permeabilization.

To study the spectrum of activity of 3 in combination with minocycline and with rifampicin, we examined in vitro potency against other clinical isolates of highly pathogenic gram-negative bacteria, including Acinetobacter baumannii, Enterobacter cloacae, and Klebsiella pneumoniae. Table 6 summarizes these results and indicates strong synergy of 3 with minocycline in other gram-negative species except AB031 and K. pneumoniae 1 10193 where the effect of 3 on minocycline is at best marginally additive. Surprisingly, combination of 3 with rifampicin displayed potent synergy in all tested isolates, with FIC indices < 0.25. The synergistic MIC of rifampicin in combination with 4 pg/mL of 3 against rifampicin-resistant E. cloacae 1 17029 was 0.063 pg/mL, which is 16-fold lower than rifampicin-susceptible breakpoints of < 1 pg/mL

EXAMPLE 4 - Time-kill curve

The kinetics of P. aeruginosa PA01 killing as a function of time, using mono- and combination-therapy of minocycline, rifampicin, and compound 3, are shown in Figures 4 and 13 Minocycline alone was not bactericidal even at 4 * MIC after 6 h, while rifampicin as a monotherapy was bactericidal at 4 ^χ MIC after 2 h of drug exposure (Figure 13). Compound 3 showed bactericidal activity at 1 ^χ MIC after 2 h, and more rapid killing was observed at 2 ^χ MIC and 4 ^χ MIC for only 30 and 10 mins antimicrobial exposure respectively (Figure 13). Minocycline, rifampicin, or 3 at sub-inhibitory concentrations were unable to suppress bacteria growth in monotherapy, even after 6 h exposure (Figure 4). However, upon combination with sub-MIC (1 /8 to 1 /2 MIC) of 3, in vitro bactericidal activities of minocycline and rifampicin were both enhanced, yielding synergistic killing at sub-MIC concentration (1 /8 ^χ MIC) after 90 mins of incubation (Figure 4).

EXAMPLE 5 - Resistance study

The ability of drug combinations to suppress resistance development was determined using wild-type P. aeruginosa PA01 . This assay was validated by demonstrating that the MIC of colistin and tobramycin increased by 1024- and 256-fold respectively over 25 serial passages, while that of minocycline increased by 16-fold (Figure 5). These results suggest that conjugate 3 reduces the likelihood of resistance acquirement relative to colistin and tobramycin. Upon combination with compound 3, the emergence of resistance in minocycline was suppressed by 4-fold while rifampicin did not promote resistance either as monotherapy or in combination with 3 (Figure 5). EXAMPLE 6 - Mode of Action Studies

Outer membrane permeabilization

Since the negatively-charged outer membrane of P. aeruginosa serves as the first barrier that prevents the uptake of antibiotics, ^{7, 8} the ability of the amphiphilic cationic compounds to perturb this lipid bilayer was investigated in PAO1 using carboxyfluorescein diacetate succinimidyl ester (CFDASE), a cell permeable dye. ²⁸ We reasoned that the amphipathic nature of the hybrids might confer membrane effects similar to those of the host defense peptides on them. The increased fluorescence induced by the compounds was calculated by subtracting the fluorescence of negative control that was treated similarly but in the absence of drug, while 1 % Triton X-100 that exhibited the highest fluorescence compared to other treatments served as the positive control (Figure 6). At the concentration tested (32 pg/mL), the outer membrane permeabilization induced by 3 was slightly lower than that of colistin. In contrast, reference compound 4 displayed the weakest membrane permeabilizing ability with the lowest fluorescence increase.

Cytoplasmic membrane depolarization

Compounds that perturb the bacterial outer membrane can potentially be trapped within the pe iplasmic space where they could interfere with the respiratory chain on the cytoplasmic membrane to induce death. Indeed, this has been proposed as one of the mechanisms by which polymyxin exerts its antibacterial effects, among many others. ²⁹ Depolarization of cytoplasmic membrane can lead to loss of membrane potential, an important electrochemical gradient used by the bacteria to maintain an active and functional efflux system. ³⁰ To investigate the effect of 3 on bacterial cytoplasmic membrane, diSC3-5, a membrane potential-dependent probe, was used to study the differential in fluorescence caused by membrane depolarization. ¹⁹ A dose-dependent fluorescence increasing was observed for all the tested antimicrobials in PAO1 . At 32 pg/mL, colistin was observed to depolarize the cytoplasmic membrane faster than other compounds in the first 300 sees, with an accompanying decrease in fluorescence thereafter (Figure 7). Although reference compound 4 displayed similar properties as colistin, the decline in fluorescence seems to be specific for colistin. Compound 3 however displayed the highest fluorescence up till 1200 sees, while only weak membrane depolarization ability was observed for tobramycin at 32 pg/rnL, a 128-fold higher value than its MIC.

Swimming motility assay

Swimming motility is a flagellum-dependent bacterial movement that is governed by the respiratory chain on the cytoplasmic membrane. When cytoplasmic membrane potential or proton motive force (PMF) is disrupted, the electron transfer across respiratory chain is inhibited, resulting in a reduction of ATP synthesis which is essential for flagellar function. ³¹ Previous studies have implicated that amphiphilic tobramycin- moxifloxacin hybrids can perturb the PMF resulting in reduced or inefficient efflux. ¹⁷ We therefore studied the effect of the hybrids 3 and 4 on the swimming motility of PA01 by monitoring its movement on low-viscosity swim plates (0.3 % agar, w/v). These studies demonstrated that compounds 3 and 4 significantly constrict the swimming motility relative to the untreated control in a dose-dependent manner (Figure 8). and the observed effects were superior to colistin at sub-inhibitory concentrations, while tobramycin was unable to inhibit bacterial motility with similar bacterial swimming diameters to untreated control.

EXAMPLE 7 - Hemolytic activity and cytotoxicity

A potential problem usually associated with membrane-active agents is their toxicity towards eukaryotic cells. The hemolytic properties of the hybrid molecules were first examined using freshly collected pig erythrocytes. All hybrids demonstrated lower hemolytic activities (< 20 %) relative to 4, which is highly toxic with 87 % hemolysis at the highest measured concentration of 512 g mL (Figure 9A). As to the structure- activity relationships (SAR) between the hybrids, a slight increase in hemolytic activity with increase in carbon length was evident. Compound 3 was also tested against human epithelial prostate (DU145) and breast (JIMT-1 ) cancer cell lines, with greater than 50 % viability at 20 μΜ (25.2 pg/mL), a six times higher concentration than effective synergistic concentration (4 Mg/mL) in combination therapy (Figure 9B). EXAMPLE 8 - In vivo efficacy

To gain insights into the potential clinical benefits of compound 3, an in vivo efficacy evaluation using Galleria mellonella infection model was initiated. The maximum tolerable dose was first determined by injecting drugs alone at high concentrations (100 or 200 mg/kg), and the survival rates scored for 4 days. As shown in Figure 14, 100% survival was observed after 4 days in the group that had been injected with 200 mg/kg of 3, indicating the relative safety of the compound to the worms at this dose. Next, the ability of the drug or drug combinations to protect larvae from MDR P. aeruginosa P262 infection was determined at single doses of 75 mg/kg in drug monotherapy or 12.5 + 12.5, 25 + 25, 37.5 + 37.5, or 75 + 75 mg/kg in drug combinations (Figure 10). 100% mortality was observed in the monotherapy of minocycline, rifampicin, and 3 at 75 mg/kg after 24 h, and in combination at lower doses. However, combinations of minocycline or rifampicin with 3 at a high dose of 75 + 75 mg/kg both resulted in 77% survival after 24 h, demonstrating the ability of this compound to offer protection against infection at very tolerable dose. Interestingly, it appears that combination therapy of rifampicin and 3 at low dosage appears to be superior when compared to combinations of minocycline and 3. This is rather surprising as the in vitro studies suggest a lower MIC for minocycline when compared to rifampicin. This discrepancy may be related to the different pharmacokinetics of minocycline and rifampicin in the larvae or the difference between bacteriostatic minocycline and bactericidal rifampicin (Figure 13).

EXAMPLE 9 - Chemical synthesis

General information

NMR spectra ( ¹ H, ¹³ C, DEPT, COSY, HSQC and HMBC) were recorded on a Bruker Avance 500 spectrometer (500 MHz for ¹ H NMR, 126 MHz for ¹³ C). All reactions were monitored by analytical thin-layer chromatography (TLC) on pre-coated silica gel plates 60 F254 (0.25 mm, Merck, Ontario, Canada), and the spots were visualized by ultraviolet light and/or by staining with ninhydrin solution in n-butanol. Mass spectrometry was carried by ESI analyses on a Varian 500 MS Ion Trap Mass Spectrometer, and MALDI-TOF on a Bruker Daltonics Ultraflex MALDI TOF/TOF Mass Spectrometer. Chromatographic separations were performed on a silica gel column by flash chromatography (Kiesel gel 40, 0.040-0.063 mm; Merck, Ontario, Canada). Yields were calculated after purification. When reactions were carried out under anhydrous conditions, the mixtures were maintained under nitrogen atmosphere. Analytical HPLC was performed on Hitachi LC system equipped with autosampler, using Superspher 100 RP-18 column and a detection wavelength of 260 nm. The purity of final compounds determined by HPLC analysis were > 95%.

General procedure for Boc-Lys(Boc)-OH

Commercially available L-lysine (0.146 g, 1.0 mmol) dissolved in H2O (5.0 mL) was mixed with NaHCO ₃ (0.252 g, 3.0 mmol). B0C2O (1 .048 g, 4.8 mmol) in 10.0 mL of THF was subsequently added to the mixture at 0 °C and stirred at room temperature overnight. At the end, THF was evaporated and the mixture was washed with diethyl ether. The aqueous layer was acidified to pH = 6 with citric acid, followed by CH2CI2 extraction. The organic layer was washed with water and brine, and dried over anhydrous Na2SO ₄ . The mixture was then concentrated and purified by flash column chromatography (eluted with CH2Cl2/MeOH from 100:0 to 15:1 , v/v) to afford the desired compound as white solid (Yield: 90 %).

General procedure for A/-dodecyl-10-aminomethyl-9-chloroanthracene hydrochloride (7) Dodecylamine 6 (0.185 g, 1 .0 mmol) and aromatic aldehyde 5 (0.289 g, 1 .2 mmol) were dissolved in dry CH2Cfe:MeOH (1 :1 , v/v) (20.0 mL) and stirred at room temperature overnight. The mixture was cooled to 0 °C, and exposed to sodium borohydride (0.1 13 g, 3.0 mmol) at room temperature overnight. The reaction mixture was subsequently concentrated, re-dispersed in diethyl ether, and treated with 2N NaOH (20.0 mL) at room temperature for additional 15 mins. The organic layer was separated from the aqueous phase, washed with water and brine, dried over anhydrous Na2SO4, concentrated, and purified by flash column chromatography (eluted with CH2Cl2/MeOH from 300:0 to 30:1 , v/v) to afford the desired compound as solid (Yield: 70 %). General procedure for Boc-Lys(Boc)-A/-dodecyl-10-Aminomethyl-9-chloroanthracene (8) Boc-Lys(Boc)-OH (0.520 g, 1 .5 mmol) dissolved in DMF:CHCI ₃ (5:2, v/v) (7.0 ml_) was activated with DIPEA (0.388 g, 3.0 mmol) and HBTU (0.569 g, 1 .5 mmol) at 0 °C for 15 mins, and treated with 7 (0.410 g, 1 .0 mmol). The mixture was stirred at 0 °C to room temperature overnight, concentrated under reduced pressure, and the resulting solution diluted in ethyl acetate. The mixture was washed with 0.5 M KHS04, water, and brine successively, and dried over anhydrous Na2S0 ₄ . The organic layer was concentrated and purified by flash column chromatography (eluted with Ch Cb/MeOH from 300:0 to 100:1 , v/v) to afford the desired compound as solid (Yield: 90 %).

General procedure for Lys-A/-dodecyl-10-Aminomethyl-9-chloroanthracene trifluoroacetate (4)

The Boc-Lys(Boc)-N-alkyl-aromatic compound 8 (0.222 g, 0.3 mmol) was dissolved in CH2Cl2:TFA (2:1 , v/v) (20.0 ml_) and stirred at room temperature for 1 h. The reaction was monitored by TLC (ChbCla/NHUOH/MeOH, 5:1 :1 ). At the end of the reaction, the mixture was evaporated to dryness, and purified by C-18 reverse-phase flash column chromatography (eluted with deionized water) to get analytically pure compound 4 (Yield: 62 %).

General procedure for 5-0-(Bromo-alkylated)-1 ,3,2',6',3"-penta-A/-(teri-butoxycarbonyl) 4',2",4",6"-tetra-OTBDMS-tobramycin (1 Oa-c)

Commercially available tobramycin was treated with Boc anhydride and TBDMSCI to give 9, as previously described. ²² 9 (1 .425 g, 1.0 mmol) was alkylated with n-dibromoalkane (5.0 mmol) in dry toluene (10.0 ml_) under phase-transfer catalytic- assisted condition of KOH (0.168 g, 3.0 mmol) and tetrabutyl ammonium bromide (0.034 g, 0.1 mmol). The mixture was stirred at room temperature overnight. At the end, solvent was evaporated under reduced pressure and the crude compound purified by flash chromatography (eluted with hexane/EtOAc from 100:0 to 100:10, v/v) to give desired product as solid (Yield: 84 % - 90 %). General procedure for 5-0-(Azido-alkylated)-1 ,3,2',6',3"-penta-A/-(feri-butoxycarbonyl)- 4' _I 2",4",6"-tetra-OTBDMS-tobramycin (11a-c)

Compounds 10a-c (1 .0 mmol) dissolved in DMF (20.0 mmol) was treated with NaN3 (0.260 g, 4.0 mmol) at 75 °C for 5 h. Water was added and the mixture was extracted thrice with EtOAc. The organic layer was washed with brine, dried over anhydrous Na2S0 ₄ , and concentrated under reduced pressure to afford the desired compound. The crude compound was subsequently purified by flash column chromatography (eluted with hexane/EtOAc from 100:0 to 80:10, v/v) (Yield: 94% - 98%).

General procedure for 5-0-(Amino-alkylated)-1 ,3,2',6',3"-penta-A/-(terf-butoxycarbonyl)- 4',2",4",6"-tetra-OTBDMS-tobramycin (12a-c)

Compounds 11a-c (1.0 mmol) dissolved in methanol (20.0 mL) was exposed to a hydrogen atmosphere with a catalytic amount of palladium hydroxide on carbon (Pd(OH)2/C) (0.014 g, 0.1 mmol). The mixture was stirred at room temperature for 3 h, followed by filtration through celite and concentration under vacuum to afford compounds 12a-c as solid (Yield: 85 % - 90 %).

General procedure for 5-O-(alkylated-10-aminomethyl-9-chloroanthracene)-1 ,3,2',6',3"- penta-A/-(teri-butoxycarbonyl)-4',2",4",6"-tetra-OTBDMS-tobr amycin (13a-c)

Compounds 12a-c (1.0 mmol) and commercially available aromatic aldehyde 5 (0.289 g, 1 .2 mmol) were dissolved in dry CH2CI ₂ :MeOH (1 :1 , v/v) (20.0 mL) and stirred at room temperature overnight. The resulting clear solution was cooled to 0 °C, sodium borohydride (0.1 13 g, 3.0 mmol) added and stirred at room temperature for 3 h. The solvents were then evaporated and the crude re-dispersed in diethyl ether followed by the addition of 2N NaOH (20.0 mL). The mixture was stirred at room temperature for 15 mins and the organic layer separated from aqueous phase, washed with water and brine, and dried over anhydrous Na2SO ₄ . The mixture was concentrated and purified by flash column chromatography (eluted with ChteC /MeOH from 300:0 to 30:1 , v/v) to give the desired product as solid (Yield: 55 % - 85 %). General procedure for secondary amide coupling (14a-c)

Boc-Lys(Boc)-OH (0.520 g, 1 .5 mmol) dissolved in DMF (30.0 mL) was activated with DIPEA (0.388 g, 3.0 mmol) and HBTU (0.569 g, 1 .5 mmol) at 0°C for 15 mins and subsequently treated with 13a-c (1.0 mmol). The mixture was stirred at 0°C to room temperature overnight. The reaction progress was monitored by TLC (CH ₂ CI ₂ /MeOH, 35:1 ), and at the end, the mixture was diluted with water and extracted with EtOAc. The organic layer was washed with water and brine, dried over anhydrous Na2S04, and concentrated. The resulting residue was purified by flash column chromatography (eluted with CH ₂ CI ₂ /MeOH from 300:0 to 30:1 , v/v) to afford the desired compound as solid (Yield: 74 % - 95 %).

General procedure for deprotection of Boc and TBDMS Groups (1-3)

Compounds 14a-c (0.2 mmol) were treated with 40 % HCI in MeOH (30.0 mL) at room temperature for 1 -3 h. The reaction progress was monitored by TLC (MeOH/ChbCb/NhUOH, 6:4:3). At the end of the reaction, the mixture was concentrated under reduced pressure to get the solid tobramycin conjugate as salt. The crude was further purified via C-18 reverse-phase flash column chromatography (eluted with deionized water) to afford analytically pure compounds (Yield: 73 % - 78 %).

EXAMPLE 10 - ¹ H & ¹³ C NMR characterization of compounds

Compound 1

Yield: 78 %. ¹ H NMR (500 MHz, Deuterium Oxide) δ 8.74 - 8.60 (m, 2H, anthracene), 8.33 - 8.24 (m, 2H, anthracene), 7.86 - 7.73 (m, 4H, anthracene), 5.99 (d, J = 15.4 Hz, 1 H, N-CH^-anthracene), 5.30 (d, J = 15.3 Hz, 1 H, N-CH H ² -anthracene), 5.14 (d, J = 2.5 Hz, 1 H, H-1 '), 4.95 (d, J = 3.4 Hz, 1 H, H-1 "), 4.43 (t, J = 6.3 Hz, 1 H, a- CH of Lys), 4.29 - 4.24 (m, 1 H, H-5'), 4.03 - 3.94 (m, 2H, H-4, H-4'), 3.83 - 3.77 (m, 2H, H-6, H-2"), 3.65 - 3.46 (m, 5H, H-1 , H-3, H-2', H-4", H-5"), 3.39 - 3.30 (m, 2H, H- 6'), 3.21 - 3.12 (m, 3H, H-5, H-3", H-6"), 3.03 - 2.95 (m, 1 H, N-CH ⁱ H ² CH ₂ ), 2.90 - 2.64 (m, 5H, N-CH H ² CH ₂ , z-CH ₂ of Lys, 0-CH ₂ of linker), 2.59 - 2.54 (m, 1 H, H-2), 2.39 - 2.32 (m, 1 H, H-6"), 2.24 - 2.12 (m, 2H, H-3'), 1 .97 - 1 .82 (m, 3H, H-2, -CH ₂ of Lys), 1 .64 - 1 .54 (m, 2H, 5-CH ₂ of Lys), 1 .45 - 1.31 (m, 2H, y-CH ₂ of Lys), 1.27 - 1.10 (m, 3H, CH ₂ of linker), 1.09 - 0.99 (m, 1 H, CH ₂ of linker). ¹³ C NMR (126 MHz, Deuterium Oxide) δ 169.34 (NCOCH), 131.18, 130.09, 128.13, 127.48, 127.40, 126.65, 125.62, 124.36, 100.79 (anomeric CH-1 "), 93.15 (anomeric CH-1 '), 82.41 , 81 .22, 77.20 (CH-4), 75.41 (CH-5'), 73.18, 72.80 (O-CH ₂ -linker), 68.44, 64.30, 63.42 (CH-6), 58.41 (CH2-6"), 54.46, 50.61 (a-CH of Lys), 49.36, 48.60, 47.65, 45.32 (N-CH2CH2), 40.74 (N-CH2- anthracene), 38.91 (ε-CHa of Lys), 38.60 (CH-6'), 30.90 (β-ΟΉ ₂ of Lys), 28.34 (CH ₂ -3'), 28.18 (CH2-2), 26.55 (5-CH ₂ of Lys), 25.36, 24.45, 21 .16; MALDI-TOF-MS: m/e calc'd for C43H ₆ 7CIN ₈ OioNa: 913.457, found: 913.463 [M+Na] ⁺ .

Compound 2

Yield: 75 %. ¹ H NMR (500 MHz, Deuterium Oxide) δ 8.49 - 8.44 (m, 2H, anthracene), 8.15 - 8.1 1 (m, 2H, anthracene), 7.71 - 7.62 (m, 4H, anthracene), 5.70 (d, J = 15.4 Hz, 1 H, N-C/W-anthracene), 5.35 - 5.28 (m, 2H, N-CH H ² -anthracene, H-1 '), 5.16 (d, J = 3.5 Hz, 1 H, H-1 "), 4.37 (t, J = 6.3 Hz, 1 H, a-CH of Lys), 4.32 - 4.27 (m, 1 H, H-5'), 4.15 (t, J = 9.8 Hz, 1 H, H-4), 3.96 - 3.89 (m, 3H, H-6, H-4', H-2"), 3.83 - 3.76 (m, 4H, H-5, O-CHiH ₂ of linker, H-4", H-5"), 3.75 - 3.72 (m, 1 H, H-6"), 3.67 - 3.55 (m, 5H, H-1 , H-3, H-2', H-3", O-CH1H2 of linker), 3.53 - 3.49 (m, 1 H, H-6"), 3.40 - 3.33 (m, 2H, H-6'), 2.98 - 2.90 (m, 1 H, N-CH ⁱ H ² CH ₂ ), 2.86 - 2.78 (m, 2H, -CH ₂ of Lys), 2.62 - 2.52 (m, 2H, H-2, N-CH ¹ H ² CH ₂ ), 2.25 - 2.20 (m, 2H, H-3'), 2.02 - 1.96 (m, 1 H, H-2), 1.92 - 1 .82 (m, 2H, β-CH? of Lys), 1 .64 - 1 .57 (m, 2H, 6-CH ₂ of Lys), 1 .44 - 1.35 (m, 3H, γ- CH of Lys, CH ₂ of linker), 1 .32 - 1 .27 (m, 1 H, CH ₂ of linker), 1 .1 1 - 1 .04 (m, 1 H, CH ₂ of linker), 1 .02 - 0.91 (m, 2H, CH ₂ of linker), 0.83 - 0.63 (m, 4H, Ctf ₂ of linker), 0.62 - 0.53 (m, 3H, CH ₂ of linker). ³ C NMR (126 MHz, Deuterium Oxide) δ 169.14 (NCOCH), 131 .04, 129.85, 127.86, 127.19, 127.09, 126.25, 125.25, 124.12, 101 .39 (anomeric CH- 1 "), 92.71 (anomeric CH-1 '), 82.04, 81.95, 76.97 (CH-4), 75.59 (CH-5'), 73.84 (O-CH2- linker), 73.18, 68.56, 64.70, 63.29, 59.12 (CH2-6"), 54.72, 50.72 (a-CH of Lys), 49.76, 48.45, 47.37, 45.80 (N-CH2CH2), 40.80 (N-CH ₂ -anthracene), 38.94 (E-CH ₂ of Lys), 38.59 (CH-6'), 30.72 (β-0½ of Lys), 29.19, 28.20, 28.15, 28.08, 28.02, 27.25, 26.49 (δ- CH ₂ of Lys), 25.15, 24.86, 21 .24; MALDI-TOF-MS calc'd for C47H ₇ 5CIN ₈ OioNa: 969.519, found: 969.523 [M+Na] ⁺ . Compound 3

Yield: 73 %. ¹ H NMR (500 MHz, Deuterium Oxide) δ 8.69 - 8.58 (m, 2H, anthracene), 8.30 - 8.24 (m, 2H, anthracene), 7.79 - 7.70 (m, 4H, anthracene), 5.87 (d, J = 15.4 Hz, 1H, N-CHW-anthracene), 5.47 (d, J= 15.3 Hz, 1H, N-CH ¹ H ² -anthracene), 5.43 (d, J = 2.6 Hz, 1H, H-1'), 5.22 (d, J = 3.6 Hz, 1H, H-1"), 4.40 (t, J = 6.3 Hz, 1H, a- CH of Lys), 4.32 - 4.28 (m, 1 H, H-5'), 4.21 (t, J = 9.8 Hz, 1 H, H-4), 3.99 - 3.74 (m, 11 H, H-5, H-6, H-2', H-4', H-2", H-4", H-5", H-6", O-CH ₂ of linker), 3.67 - 3.57 (m, 3H, H-1 , H- 3, H-3"), 3.45 - 3.32 (m, 2H, H-6'), 3.03 - 2.96 (m, 1H, N-C/WCH ), 2.89 - 2.79 (m, 2H, z-CH ₂ of Lys), 2.78 - 2.71 (m, 1H, N-CH ¹ H ² CH ₂ ), 2.58 - 2.54 (m, 1H, H-2), 2.32 - 2.23 (m, 2H, H-3'), 2.03 - 1.86 (m, 3H, H-2, β-ΟΗ ₂ of Lys), 1.75 - 1.66 (m, 2H, CH ₂ of linker), 1.65 - 1.59 (m, 2H, 6-CH ₂ of Lys), 1.45 - 1.29 (m, 6H, y-CH ₂ of Lys, CH ₂ of linker), 1.25- 1.18 (m, 2H, CH ₂ of linker), 1.13- 0.94 (m, 4H, CH ₂ of linker), 0.91 -0.83 (m, 2H, CH ₂ of linker), 0.78 - 0.71 (m, 1H, CH ₂ of linker), 0.68 - 0.59 (m, 3H, CH of linker), 0.58 - 0.50 (m, 2H, CH ₂ of linker). ¹³ C NMR (126 MHz, Deuterium Oxide) δ 169.21 (NCOCH), 131.30, 130.11, 128.10, 127.30, 127.18, 126.53, 125.43, 124.24, 101.39 (anomeric CH-1"), 92.73 (anomeric CH-1'), 81.93, 81.89, 76.82 (CH-4), 75.66 (CH-5'), 73.84 (O-CH ₂ -linker), 73.22, 68.54, 64.77, 63.24, 59.27 (CH2-6"), 54.76, 50.73 (a-CH of Lys), 49.79, 48.44, 47.34, 46.04 (N-CH2CH2), 41.03 (N-CH ₂ -anthracene), 38.94 (£-CH ₂ of Lys), 38.55 (CH-6'), 30.74 ( -CH ₂ of Lys), 29.57 (CH2 of linker), 29.13, 29.00, 28.70, 28.56, 28.19, 28.16 (CH ₂ -3'), 27.98, 27.91 (CH ₂ -2), 27.32, 26.50 (6-CH ₂ of Lys), 25.40, 25.22, 21.28; MALDI-TOF-MS: m/e calc'd for CsiHssCINsOioNa: 1025.582, found: 1025.586 [M+Na] ⁺ .

Compound 4

Yield: 62 %. H NMR (500 MHz, Methanol^) δ 8.61 - 8.57 (m, 2H, anthracene), 8.43 - 8.38 (m, 2H, anthracene), 7.69 - 7.61 (m, 4H, anthracene), 6.10 (d, J = 15.3 Hz, 1H, N-CHW-anthracene), 5.45 (d, J = 15.3 Hz, 1H, N-CH ¹ H ² -anthracene), 4.28 (t, J = 6.2 Hz, 1 H, a-CH of Lys), 3.02 - 2.96 (m, 1 H, N-CH ⁱ H ² CH ₂ ), 2.78 - 2.69 (m, 2H, z-CH ₂ of Lys), 2.68 - 2.60 (m, 1H, N-CH H ² CH ₂ ), 1.86 - 1.76 (m, 2H, p-CH ₂ of Lys), 1.59 - 1.52 (m, 2H, 5-CH ₂ of Lys), 1.48- 1.39 (m, 2H, y-CH ₂ of Lys), 1.34- 1.20 (m, 10H, N- CH ₂ (CH)ioCH ₃ ), 1.17 - 1.11 (m, 2H, N-CH ₂ (CH ₂ )ioCH ₃ ), 1.09 - 1.03 (m, 2H, N- CH2(CH ₂ )ioCH3), 0.97 - 0.77 (m, 9H, N-CH ₂ (CH ₂ )ioCH ₃ ). ³ C NMR (126 MHz, Methanol-c 4) δ 168.34 (NCOCH), 131 .61 , 130.03, 128.40, 127.33, 126.75, 126.61 , 125.23, 124.19, 50.46 (a-CH of Lys), 45.08 (N-CH2CH2), 39.48 (N-CH ₂ -anthracene), 38.70 (ε-CHa of Lys), 31 .64, 30.92 (β-CHa of Lys), 29.27, 29.16, 29.06, 29.02, 28.82, 28.56, 28.37, 26.76 (5-CH ₂ of Lys), 25.95, 22.31 , 21 .25, 13.01 (CH ₂ CH ₃ ); MALDI-TOF- MS: m/e calc'd for C33H49CIN3O: 538.356, found: 538.358 [M+H] ⁺ .

EXAMPLE 11 - Biological activity assays

Bacterial isolates

Bacterial isolates were obtained as part of the Canadian National Intensive Care Unit (CAN-ICU) study ⁶¹ and Canadian Ward Surveillance (CANWARD) studies ⁶² ' ⁶³ . The CAN-ICU study included 19 medical centres across Canada with active ICUs. From September 2005 to June 2006, 4180 isolates represented in 2580 ICU patients were recovered from clinical specimens including blood, urine, wound/tissue, and respiratory specimens (one pathogen per cultured site per patient). Only "clinically significant" specimens from patients with a presumed infectious disease were collected. The isolates obtained were shipped to the reference laboratory (Health Sciences Centre, Winnipeg, Canada) on Amies charcoal swabs. Then isolates were sub-cultured onto appropriate medium and stocked in skim milk at -80 °C until subsequent MIC testing was carried out. The quality control strains including Staphylococcus aureus ATCC 29213, methicillin-resistant S. aureus (MRSA) ATCC 33592, Enterococcus faecalis ATCC 29212, Enterococcus faecium ATCC 27270, Streptococcus pneumoniae ATCC 49619, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Klebsiella pneumoniae ATCC 13883 were acquired from the American Type Culture Collection (ATCC). The clinical strains, including methicillin-resistant Staphylococcus epidermidis (MRSE), CAN-ICU 61589 (cefazolin MIC > 32 pg/mL), gentamicin resistant E. coli CAN-ICU 6 714, Amikacin-resistant E. coli CAN-ICU 63074 (MIC = 32 pg/mL), gentamicin resistant P. aeruginosa CAN-ICU 62584, Strenotrophomonas maltophilia CAN-ICU 62584, and Acinetobacter baumannii CAN-ICU 63169 were obtained from hospitals across Canada as a part of the CAN-ICU study. Methicillin-susceptible S. epidermidis (MSSE) CANWARD-2008 81388 was obtained from the 2008 CANWARD study, while gentamicin-resistant tobramycin-resistant ciprofloxacin-resistant [aminoglycoside modifying enzyme aac(3)-lla present] E. coli CANWARD-2011 97615, and gentamicin-resistant tobramycin-resistant P. aeruginosa CANWARD-201 1 96846 were obtained from the 201 1 CANWARD study. In addition, P. aeruginosa PA01 , P. aeruginosa P259-96918, P. aeruginosa P262-101856, P. aeruginosa P264-104354, colistin-resistant P. aeruginosa 91433, colistin-resistant P. aeruginosa 101243, A. baumannii AB027, A. baumannii AB030, A. baumannii AB031 , A. baumannii 1 10193, Enterobacter cloacae 1 17029, and Klebsiella pneumonia 1 16381 were kindly provided by Dr. George G. Zhanel. The efflux pump-mutated strains, P. aeruginosa PAO200 and P. aeruginosa PAO750, were provided by Dr. Ayush Kumar from University of Manitoba in Canada.

Multi-drug resistance in P. aeruginosa was defined as concomitant resistance to 3 or more chemically unrelated antimicrobial classes, while extremely drug resistant was defined as concomitant resistance to 5 or more chemically unrelated antimicrobial classes.

Antimicrobial activity assay

The antimicrobial activity of the compounds against a panel of bacteria was evaluated by microliter dilution method in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. The overnight bacterial culture was diluted in saline to 0.5 McFarland turbidity, and then 1 :50 diluted in Mueller-Hinton broth (MHB) for inoculation. The antimicrobial agents were 2-fold serially diluted in MHB in 96-well plate and incubated with equal volumes of inoculum for 18 h at 37 °C. The lowest concentration that prevented visible bacterial growth was taken as the MIC for each antimicrobial agent. The broth with or without bacterial cells was employed as positive or negative controls, respectively.

Combination studies with different antibiotics

FIC index was determined by setting up standard checkerboard assay in 96-well plate as previously described. ⁶⁴ Each antibiotic to be tested was serially diluted along the abscissa in MHB, while adjuvant was diluted along the ordinate to create a 10 χ 7 matrix. The bacterial culture was prepared in MHB by 1 :50 dilution from the 0.5 McFarland turbidity culture in saline. The inoculum was added to each well of the plate and incubated for 18 h at 37 °C. After the incubation, plates were read on EMax ^® Plus microplate reader (Molecular Devices, Sunnyvale, CA, USA). MIC was recorded as wells with the lowest concentration of drugs with no bacterial growth. The FIC for each antibiotic was calculated as the concentration of the antibiotic for a well showing no growth in the presence of adjuvant divided by the MIC for that antibiotic alone. The FIC for each adjuvant was calculated as the concentration of the adjuvant for a well showing no growth in the presence of antibiotic divided by the MIC for that adjuvant alone. The FIC index is the sum of the two FICs. Chemical-chemical interactions with an FIC index < 0.5 were deemed synergistic; 0.5 - 4, no interaction; and > 4, antagonism.

Time-kill curve assay

The kinetics of bacterial killing was measured using P. aeruginosa PA01 , as previously described. ⁵⁹ Overnight bacterial culture was diluted in saline to 0.5 McFarland turbidity and then 1 :50 diluted in Luria-Bertani (LB) broth. The cell suspension was incubated with minocycline, rifampicin, or hybrid 3 diluted in PBS (pH 7.2) alone at desired concentrations (1/2 χ , 1 χ, 2 χ, 4 ^χ MIC). For synergistic time-kill, the combination of compound 3 with minocycline or rifampicin at various concentrations, 1/8 + 1/8, 1/8 + 1/4, 1/4 + 1/4, 1/2 + 1/4, and 1/2 + 1/2 ^χ MIC, were determined. Samples were incubated at 37 °C for 6 h. At specific intervals (0, 10, 30, 60, 90, 120, 240, and 360 mins), aliquots (50 μΐ_) were removed from the samples, serially diluted in PBS and plated on LB agar plates. Bacterial colonies were formed and counted after 20 h of incubation at 37 °C.

Resistance development assay

Wild-type P. aeruginosa PAO1 was used to study resistance development against antibiotics by sequential passaging method as previously described. ⁶⁵ Briefly, MIC testing was first conducted for all drugs or drug combinations to be tested, as described above. After 18 h incubation, the bacterial cells growing in the well of half- MIC concentration were harvested and diluted to 0.5 McFarland in saline followed by 1 :50 dilution in fresh MHB broth. The inoculum was subjected to next passage MIC testing, and the process repeated for 25 passages. The fold change in MIC was plotted against the number of passages.

Outer membrane permeability assay

The CFDASE dye was used to determine the outer membrane permeability of drugs against P. aeruginosa PAO1 , following established protocols. ²⁸ Logarithmic phase P. aeruginosa was harvested by centrifugation and washed twice with PBS. The bacterial cells were resuspended in the same buffer to OD600 of 0.5, followed by staining with CFDASE at 100 μΜ for 30 mins at 37°C. The unbound dye was then removed by washing the cells with excess buffer, and the cells were again resuspended to the initial volume. The bacterial suspension was treated with drugs at 37 °C for 30 mins at desired concentration and the supernatant obtained by centrifugation was transferred to 96-well black plate for measuring the fluorescence at an excitation wavelength of 488 nm and an emission wavelength of 520 nm using a microplate reader FlexStation 3 (Molecular Devices, Sunnyvale, CA, USA).

Cytoplasmic membrane depolarization assay

To assess the effect of the compounds on cytoplasmic membrane potential, diSC3-5, the membrane-potential-sensitive fluorescent dye was utilized to determine the membrane depolarization of P. aeruginosa PAO1 as previously described. ¹⁹ Overnight growth P. aeruginosa PAO1 was diluted in fresh LB broth and cultured to the mid-log phase. The bacterial cells were harvested and washed three times with 5 mM sodium HEPES buffer, pH 7.4, containing 20 mM glucose, and resuspended to Οϋβοο of 0.05 in the same buffer. The cell suspension was incubated with 0.2 mM EDTA and 0.4 μΜ diSC3-5 in the dark for 2 h at 37 °C under constant shaking (150 rpm). 100 mM KCI was then added to equilibrate the cytoplasmic and external K ⁺ , and incubated for additional 30 mins. The depolarization assay was carried out in 96-well black plate by adding the antimicrobial agents to 100 pL of the above cell suspension to desired concentration. Fluorescence was monitored using a FlexStation 3 (Molecular Devices, Sunnyvale, CA, USA) microplate reader at an excitation wavelength of 622 nm and an emission wavelength of 670 nm. Motility assay

Cell motility assay was performed on 0.3 % (w/v) agar media supplemented with tryptone (5 g/L) and NaCI (2.5 g/L). ⁶⁶ Antimicrobial agents were added to 25 ml_ medium to the desired concentration and poured on 100 ^χ 15 mm petri dishes followed by 2 h drying. Overnight P. aeruginosa PAO1 culture was diluted in 0.85 % saline to 1 .0 McFarland and point inoculated into the center of the motility agar plates. Plates were incubated at 37 °C for 20 h. The images presented were taken using a FluroChem®Q (Cell biosciences).

Quantification of hemolytic activity

The hemolytic activity of the compounds was determined as the amount of hemoglobin released by lysing pig erythrocytes. ⁶⁷ Fresh pig blood (provided by Dr. Charles M. Nyachoti from University of Manitoba) drawn from pig antecubital were centrifuged at 1 ,000 ^χ g for 5 mins at 4 °C, washed with PBS thrice, and resuspended in the same buffer. Compounds were 2-fold serially diluted in PBS in 96-well plate and mixed with equal volumes of erythrocyte solution. After 1 h incubation at 37 °C, intact erythrocytes were pelleted by centrifuging at 1 ,000 ^χ g for 5 mins at 4 °C, and the supernatant was transferred to a new 96-well plate. The hemoglobin release was monitored at 570 nm using an EMax ^® Plus microplate reader (Molecular Devices, Sunnyvale, CA, USA). Blood cells in PBS and 0.1 % Triton X-100 were employed as negative and positive controls respectively.

Cytotoxicity assay

DU145 (ATCC, Manassas, VA, USA) and JIMT-1 (DSMZ, Braunschweig, Germany) were cultured and maintained in Dulbecco's modified Eagle's References medium supplemented with 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 10 % (v/v) fetal bovine serum (FBS) at 37 °C under a humidified atmosphere of 5 % CO2 and 95 % air. The methanethiosulfonate (MTS) cell viability assay was employed to measure the cytotoxicity of compound 3 as previously described. ¹⁶ Briefly, the cells were seeded in 96-well plate with a final concentration of 7500-9000 cells per well and incubated for 24 h. Then the cells were treated with test compound at final concentrations of 2.5 to 30 μΜ and incubated for an additional 48 h at the same condition. MTS reagent (20 %, v/v) was further added to each well and the plates were incubated for 4 h on a Nutating mixer in the incubator. The optical density was measured using a SpectraMax M2 plate reader (Molecular Devices, Sunnyvale, CA, USA) at 490 nm. Only medium without cells were served as blank and the blank values were subtracted from each sample value. The cell viability relative to the control with vehicle was calculated.

Galleria mellonella model of P. aeruginosa infection

Galleria mellonella waxworms were obtained from The Worm Lady ^® Live Feeder Insects. Worms (average weight at 250 mg) were used within 7 days of delivery to determine the survival rate after bacteria or antimicrobials injection using previously described methods. ⁶⁸ The tolerability study was performed by only injecting antimicrobial agent into the worms at 100 and 200 mg/kg without bacteria. The worms (ten larvae in each group) were incubated at 37 °C and monitored for 96 h for survival. For therapeutic study, overnight XDR P. aeruginosa P262 culture was diluted in PBS to a final concentration of 1 .0 ^χ 10 ³ CFU/mL. 15 larvae per group were infected with 10 pL bacterial suspensions. After 2 h bacterial challenge, worms in monotherapy experimental groups received a 10 pL injection of minocycline, rifampicin, or compound 3 individually at 75 mg/kg. For combination groups, 3 plus minocycline and 3 plus rifampicin were injected to give final dosages of 12.5 + 12.5, 25 + 25, 37.5 + 37.5, and 75 + 75 mg/kg respectively. Only vehicle (PBS) without antimicrobials was injected as control group. The larvae were monitored for 24 h at 37 °C in petri dishes lined with filter paper and scored for survivability. Larvae considered dead if they do not respond to touch.

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Table 1. MIC of compounds against a panel of gram-positive and gram-negative bacteria

MIC ^a (pig/mL)

Organisms

Amphiphilic tobramycin-lysine conjugates

Tobramycin

1

Gram-positive bacteria

S. aureus ATCC 29213 <0.25 ^b 32 64 16

MRSA ATCC 33592 <0.25 ^b 64 64 16

MSSE CANWARD-2008 81388 <0.25 16 2

MRSE CAN-ICU 61589 (CAZ >32) l ^b 32 4

E. faecalis ATCC 29212 8 ^b 32 >128 64 16

E. faecium ATCC 27270 8 8 >128 16 8

S. pneumoniae ATCC 49619 2 ^b 32 > 128 128 32

Gram-negative bacteria

E.coli ATCC 25922 0.5 ^b 64 32 16 32

E.coli CAN-ICU 61714 (GEN-R) 8 ^b 128 64 32 32

E.coli CAN-ICU 63074 (AMK 32) 8 ^b 64 64 128 16

E.coli CANWARD-201 1 97615 (GEN-R, TOB-R,

128 ^b 64

CIP-R) aac(3')iia 64 32 32

P. aeruginosa ATCC 27853 0.5 ^b > 128 > 128 > 128 32

P. aeruginosa CAN-ICU 62308 (GEN-R) 16 ^b 128 64 16 16

P. aeruginosa CANWARD-201 1 96846 (GEN-R,

256

TOB-R) > 128 >128 64 32

S. maltophilia CAN-ICU 62584 >512 ^b >128 >128 >128 >128 A. baumannii CAN-ICU 63169 32 ^b 128 > 128 > 128 128 K. pneumoniae ATCC 13883 <0.25 ^b >128 128 >128 128

" Minimum inhibitory concentration (MIC) was determined as the lowest concentration of compound that inhibited bacteria growth. ^b MIC data of tobramycin as previously reported. ¹⁶

MRSA: Methicillin-resistant S. aureus; MSSE: Methicillin-susceptible S. epidermidis; MRSE: Methicillin-resistant S. epidermidis; CANWARD: Canadian Ward surveillance; CAN-ICU: Canadian National Intensive Care Unit surveillance; CAZ: Ceftazidime; GEN-R: Gentamicin-resistant; AMK: Amikacin; TOB-R: Tobramycin-resistant; CIP-R: Ciprofloxacin-resistant.

Table 2. MIC of compounds 1 - 4 and antibiotics against wild-type and clinical isolate P. aeruginosa.

MIC (Mg/mL)

P. aeruginosa strains Amphiphilic tobramycin-lysine conjugates Antibiotics

TOB 4

1 2 3 MOX CIP iN RMP CAZ CAM ERY TMP COL

PAOl 0.25 256 >512 128 32 1 ND 8 16 2 64 256 256 1

100036 64 256 >512 512 32 128 64 32 16 8 >512 256 256 2

101885 0.25 128 256 64 16 64 32 32 16 8 512 256 >512 0.5

P259-96918 128 64 >512 5 12 64 512 128 32 16 5 12 512 256 512 0.5

P262-101856 512 128 >512 128 32 128 32 256 1024 16 1024 1024 >1024 2

P264-104354 128 128 >512 256 32 128 32 64 16 64 1024 256 256 4

91433 8 256 32 8 8 8 2 64 16 256 16 5 12 512 4

101243 256 64 256 32 16 4 2 4 8 64 4 1024 1024 > 1024

TOB = Tobramycin; MOX = Moxifloxacin; CIP = Ciprofloxacin; MIN = Minocycline; RMP = Rifampicin; CAZ = Ceftazidime; CAM = Chloramphenicol; ERY = Erythromycin; TMP = Trimethoprim; COL = Colistin. ND = Not determined.

Table 3. Combination study of compound 3 with antibiotics against wild-type P. aeruginosa PAOl .

MIC alone Synergistic MIC MIC alone Synergistic MIC

Antibiotics FIC antibiotic Hybrid FIC hybrid FIC index

(Hg/mL) (Hg/mL) ^g/mL) ^g/mL)

Moxifloxacin 1 0.25 0.25 3 32 2 0.063 0.313

Novobiocin 1024 8 0.008 3 32 2 0.063 0.071

Minocycline 8 0.5 0.063 3 32 1 0.031 0.094

Rifampicin 16 1 0.063 3 32 1 0.031 0.094

Ceftazidime 2 1 0.5 3 32 2 0.063 0.563

Chloramphenicol 64 2 0.03 1 3 32 4 0.125 0.156

Erythromycin 256 8 0.031 3 32 4 0. 125 0.156

Trimethoprim 256 16 0.063 3 32 2 0.063 0.126

Colistin 1 1 1 3 32 1 0.031 1.031

Gentamicin 1 1 1 3 32 4 0.125 1.125

Kanamycin A 64 64 1 3 32 0.5 0.016 1.016

Amikacin 1 1 1 3 32 2 0.063 1.063

Meropenem 0.5 1 2 3 32 1 0.031 2.031

Vancomycin >1024 128 <0. 125 3 32 4 0.125 0.125 < 0.25

Table 4. Synergistic effects of compound 3 with minocycline or rifampicin against clinical MDR or XDR P. aeruginosa isolates.

P. aeruginosa

Antibiotics (MIC ^a ) FIC Hybrid (MIC) FIC FIC index Absolute MIC ^b Potentiation ⁰ strain

100036 inocycline (32) 0.063 3 (32) 0.03 1 0.094 1 32-fold

100036 Rifampicin (16) 0.016 3 (32) 0.063 0.079 0.125 128-fold

101885 Minocycline (32) 0.125 3 (16) 0.031 0. 156 2 16-fold

101885 Rifampicin (16) 0.063 3 (16) 0.031 0.094 0.125 128-fold

P259-96918 Minocycline (32) 0.031 3 (64) 0.016 0.047 0.5 64-fold

P259-9691 8 Rifampicin (16) 0.008 3 (64) 0.031 0.039 0.063 256-fold

P262- 101856 Minocycline (256) 0.008 3 (32) 0. 125 0.133 2 128-fold

P262-101856 Rifampicin ( 1024) 0.016 3 (32) 0.125 0. 141 16 64-fold

P264- 104354 Minocycline (64) 0.016 3 (32) 0.063 0.079 0.5 128-fold

P264- 104354 Rifampicin (16) 0.031 3 (32) 0.063 0.094 0.25 64-fold

91433 Minocycline (64) 0.016 3 (8) 0.25 0.266 0.5 128-fold

91433 Rifampicin ( 16) 0.25 3 (8) 0.25 0.5 2 8-fold

101243 Minocycline (4) 0.25 3 ( 16) 0.031 0.281 1 4-fold

101243 Rifampicin (8) 0.063 3 ( 16) 0.125 0.188 0.25 32-fold

" All MIC data presented in μg/mL. ^b Absolute MIC of antibiotic was determined in the presence of hybrid at 4 ng/mL. ^c Antibiotic activity potentiation at 4 Hg/mL of hybrid.

Table 5. Synergistic effects of compound 3 with minocycline and with rifampicin against P. aeruginosa

PAOl and efflux pump deficient P. aeruginosa PAO200 and PAO750 strains.

P. aeruginosa strain Antibiotic (MIC ^ig/mL) Adjuvant (MIC g/mL) FIC index

PAO l Minocycline (8) 3 (32) 0.094

PAOl Rifampicin (16) 3 (32) 0.094

PAO200 Minocycline (1) 3 (16) 0.531

PAO200 Rifampicin (8) 3 (16) 0.129

PAO750 Minocycline (1) 3 (8) 0.75

PAO750 Rifampicin (8) 3 (8) 0. 156

Table 6. Combination study of compound 3 with minocycline or rifampicin against MDR Acinetobacter baumannii, Enterobacter cloacae and Klebsiella pneumoniae.

MIC alone Synergistic MIC alone Synergistic

Organisms* Antibiotics FiC antibiotic Hybrids hybrid FIC index

(Hg/mL) MIC (ng/mL) (Hg mL) MIC (ng FIC

/mL)

AB027 Minocycline 1 0.25 0.25 3 128 1 0.008 0.258

AB027 Rifampicin 2 0.031 0.016 3 128 8 0.063 0.079

AB030 Minocycline 4 0.5 0.125 3 64 16 0.25 0.375

AB030 Rifampicin >256 32 <0. 1 5 3 64 8 0.125 0.125 < 0.25

AB031 Minocycline 2 1 0.5 3 16 2 0.125 0.625

AB031 Rifampicin 2 0.125 0.06 3 32 1 0.03 0.09

1 10193 Minocycline 2 1 0.5 3 16 2 0.13 0.63

1 10193 Rifampicin 2 0.063 0.032 3 16 1 0.063 0.095

1 17029 Minocycline 128 4 0.03 1 3 256 8 0.031 0.062

1 17029 Rifampicin 16 0.063 0.004 3 256 4 0.016 0.02

1 16381 Minocycline 128 32 0.25 3 256 16 0.063 0.313

1 16381 Rifampicin 1024 32 0.031 3 256 4 0.016 0.047

"AB027, "AB030, "AB031, and "110193 = Acinetobacter baumannii; "117029 = Enterobacter cloacae; "116381 = Klebsiella pneumoniae.

Table 7. Synergistic effects comparison of tobramycin, compound 4, and hybrids 1 - 3 with minocycline or rifampicin against P. aeruginosa PAOl .

P. aeruginosa MIC alone Synergistic IC alone Synergistic

Antibiotics FIC antibiotic Adjuvant FIC hybrid FIC index strain (pg/mL) MIC (pg/mL) (pg/mL) MIC (pg/mL)

PAOl Minocycline 8 8 1 Tobramycin 0.25 0.016 0.064 1 .064

PAOl Minocycline 8 8 1 4 256 1 0.004 1.004

PAOl Minocycline 8 4 0.5 1 >256 1 <0.004 0.5< ; <0.504

PAOl Minocycline 8 0.5 0.063 2 128 1 0.008 0.071

PAO l Minocycline 8 0.5 0.063 3 32 0.031 0.094

PAOl Rifampicin 16 8 0.5 Tobramycin 0.25 0.125 0.5 1

PAO l Rifampicin 16 8 0.5 4 256 0.004 0.504

PAO l Rifampicin 16 8 0.5 1 >256 <0.004 0.5< ; <0.504

PAOl Rifampicin 16 1 0.063 2 128 0.008 0.071

PAOl Rifampicin 16 1 0.063 3 32 1 0.031 0.094