Pyrrolobenzodiazepine dimers: novel sequence-selective, DNA-interactive, cross-linking agents with activity against Gram-positive bacteria

Tsveta Hadjivassileva, David E. Thurston and Peter W. Taylor*

School of Pharmacy, 29–39 Brunswick Square, London WC1N 1AX, UK


* Corresponding author. Tel/Fax: +44-20-7753-5867; E-mail: peter.taylor{at}ulsop.ac.uk

Received 31 March 2005; returned 17 June 2005; revised 21 June 2005; accepted 23 June 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers are synthetic sequence-selective interstrand DNA minor-groove cross-linking agents developed from anthramycins. We investigated the antibacterial activity of three dimers, SJG-136, DRG-16 and ELB-21, which differ in the structure of the PBD monomeric unit and the length of the linker region between the two identical PBD monomers.

Methods: MICs were determined against 38 methicillin-resistant Staphylococcus aureus (MRSA), 20 vancomycin-resistant enterococci (VRE), 12 isolates of Streptococcus pyogenes, 12 of Streptococcus agalactiae, 12 of Listeria monocytogenes and 24 Gram-negative clinical isolates. Binding to double-stranded DNA was assessed by determination of the DNA melting temperature (Tm).

Results: MIC90 values for SJG-136 were 0.5 mg/L against MRSA, VRE and L. monocytogenes, 0.06 mg/L against S. pyogenes and 0.03 mg/L against S. agalactiae; these were below the maximum tolerated dose of the drug. MIC90s for DRG-16 were 0.125, >0.5, 0.125, 0.015 and <0.008 mg/L, respectively. The most potent compound was ELB-21, with corresponding MIC90 values of 0.03, 0.06, 0.06, 0.015 and 0.015 mg/L. There was little or no variation in sensitivity amongst isolates from any one species. All Gram-negative species (Acinetobacter, Pseudomonas, Klebsiella, Proteus spp.) were not susceptible due to the barrier function of the outer membrane. PBD dimers showed bactericidal activity against MRSA and VRE and there was a significant post-antibiotic effect (1.5–3.5 h). Incubation of EMRSA-16 genomic DNA (50 µM) with 20 µM ELB-21 resulted in a large increase in Tm suggesting that PBD dimers exert their antibacterial effect by cross-linking of the two DNA strands.

Conclusions: These data indicate that this novel class of antibacterial agents warrants further investigation as potential antibiotics for the treatment of severe infections caused by Gram-positive pathogens.

Keywords: DNA cross-linking agents , pyrrolobenzodiazepine dimers , Staphylococcus aureus , vancomycin-resistant enterococci , MRSA


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Over the last two decades, drug-resistant Gram-positive pathogens, particularly methicillin-resistant Staphylococcus aureus (MRSA), multiresistant coagulase-negative staphylococci, penicillin-resistant Streptococcus pneumoniae and vancomycin-resistant enterococci (VRE), have emerged worldwide. Although pan-resistant Gram-positive isolates have yet to appear, MRSA and VRE infections in particular are becoming more difficult to treat.1 The recent introduction of novel antibacterial agents such as quinupristin/dalfopristin (type B/type A streptogramins) and linezolid (an oxazolidinone) has provided efficacious and safe treatment options for MRSA and VRE infections, but there remains an urgent need for new therapies, particularly as some VRE strains are already showing decreased susceptibility to these agents.2,3 As therapeutic options decrease, there has been recent interest in the development of agents that exert their antibacterial effects by binding selectively to specific sequences of duplex DNA. For example, small molecules derived from polyheterocyclic antibiotics based on distamycin have emerged recently as candidates for clinical development.4,5 These molecules are active against Gram-positive isolates such as streptococci, staphylococci and enterococci and exert their bactericidal effects by binding within the minor groove of DNA at A:T-rich sites commonly found in bacterial promoters and origins of replication. Their in vitro potencies are comparable to vancomycin and linezolid, they show efficacy against MRSA in murine models of acute infection and they display no genotoxicity.5

The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a family of DNA-interactive antibiotics derived from various Streptomyces species and examples include antitumour agents such as DC-81 and tomaymycin.6 They exert their biological activity through covalent binding to the C2-NH2 of guanine within the minor groove of DNA (Figure 1a). PBD monomers span three DNA base pairs with a preference for Pu–G–Pu (where Pu = purine; G = guanine) sequences and block transcription by inhibiting RNA polymerase activity in a sequence-specific manner.7 The potency, DNA-binding affinity and sequence specificity of PBDs have been markedly improved by linking two PBD units through their C8/C8'' positions to form a PBD dimer that can cross-link appropriately separated guanines on opposite DNA strands.8 PBD units tethered through a three-carbon diether linkage are able to bind selectively to regions of DNA that are 6–7 bp in length. For example, the PBD dimer SJG-136 (Figure 1b) predominantly binds covalently to embedded 5'-Pu-GATC-Py sequences by cross-linking opposite-strand guanines separated by 2 bp, with little or no disruption of DNA secondary structure.8,9 Sequence-selective recognition can be further enhanced by extension of the linker joining the two PBD monomers. Thus, use of a five-carbon diether linker (Figure 1) facilitates selective binding to include an additional base pair and effects cross-linking of 5'-Pu-GATTC-Py and 5'-Pu-GAATC-Py sequences.8 In this study, we report the in vitro antibacterial activity of the three PBD dimers SJG-136, DRG-16 and ELB-21.



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Figure 1. PBDs bind covalently to duplex DNA: (a) mechanism of binding via N10-C11 imine/carbinolamine moiety to the C2-amino position of a guanine residue within the minor groove of DNA. (b) Tethering of two identical PBD units through a C8/C8 diether linker improves DNA-binding efficiency.

 

    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials

PBD dimers SJG-136, DRG-16 and ELB-21 (Figure 1) were designed and synthesized according to previously described procedures.10,11 Paper discs impregnated with oxacillin, vancomycin, trimethoprim, amikacin, gentamicin, tetracycline, rifampicin, ciprofloxacin, erythromycin and clindamycin were used to determine bacterial susceptibility to these antibiotics and were purchased from Oxoid (Basingstoke, UK).

Bacterial strains

The international collection of 38 MRSA clinical isolates12 used in this study was assembled at the Royal Free and University College Medical School (RFUCMS), London. The 20 VRE, 12 Streptococcus pyogenes, 12 Streptococcus agalactiae, 12 Listeria monocytogenes, 6 Klebsiella spp., 6 Pseudomonas aeruginosa, 6 Acinetobacter spp., and 6 Proteus mirabilis strains were isolated from clinical specimens at the RFUCMS and were the gift of Professor Jeremy Hamilton-Miller. Mycobacterium aurum, M. smegmatis, M. fortuitum, M. abscessus, and M. phlei were obtained from previously described sources.13

MIC assays

In vitro susceptibilities were determined by the NCCLS broth microplate assay; 0.1 mL of dilutions of the test compound in Mueller–Hinton broth (Oxoid) were added to 0.1 mL aliquots of bacterial suspension to give a final bacterial count of 105 cfu/mL. MIC determinations for mycobacterial strains were performed as previously described.13 PBD dimers were dissolved in methanol prior to dilution in broth; appropriate concentrations of methanol were incorporated into controls to ensure that the low concentrations of solvent used had no effect on the growth of test strains.

Kinetics of bacterial killing

Mid-logarithmic or stationary phase bacteria in Mueller–Hinton broth (106 cfu/mL) were exposed to appropriate concentrations (MIC; MIC x2; MIC x4) of PBD dimers and viable counts performed at various time points following incubation with agitation (200 orbits per min on an orbital shaker) at 37°C. Experiments were carried out in duplicate.

Electron microscopy

Negatively stained images were obtained as described previously.14

Post-antibiotic effect (PAE)

Mid-logarithmic phase bacteria growing in Mueller–Hinton broth were incubated at 37°C in the presence of various concentrations of the PBD dimer for 1 h. The agent was removed by centrifugation and the cells washed with pre-warmed PBS. The pellet was suspended in pre-warmed Mueller–Hinton broth, incubated at 37°C and bacterial numbers determined over a 24 h period by viable counting. The PAE was calculated by the standard equation T–C, where T is the time taken for the viable count of the compound-treated culture to increase 10-fold after removal of the agent and C is the time taken for the untreated control to increase 10-fold under the same conditions.

Melting curves of duplex DNA

Genomic DNA was prepared from MRSA strains by guanidium thiocyanate extraction according to Pitcher et al.15 The DNA was washed with ethanol and dissolved in 10 mM Na2HPO4 containing 1 mM EDTA (pH 7.0) to give a concentration of 50 µM. Melting curves were determined spectrophotometrically ({lambda}260) in a heated chamber with a controlled increase in temperature of 1°C/min over the range 40–98°C. Optical data were imported into the Origin 5 computer package (MicroCal Inc, Northampton, MA, USA) for analysis and the melting temperature Tm determined at the midpoint of the normalized melting profiles using a published procedure.16 PBD dimers (20 µM) were added to the DNA and incubated at 37°C for 1 h prior to determination of the melting profile.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In vitro antibacterial activity

We determined the antibacterial activity of three PBD dimers (SJG-136, DRG-16 and ELB-21) with differing potencies and sequence selectivities against 94 Gram-positive isolates, 30 Gram-negative isolates and five atypical mycobacteria (Table 1). All Gram-negative isolates were insensitive to all three dimers, with MIC90 values greater than 16 mg/mL; strains could be sensitized by perturbation of the outer membrane with 1 mM EDTA, indicating that this structure precluded the agents from entering the cell. In contrast, all Gram-positive isolates were susceptible to the three dimers with MIC90 values below 1 mg/L in all cases. The most potent compound was ELB-21, with MIC90 values of 0.03 mg/L for MRSA and 0.06 mg/L for VRE. Overall, DRG-16 proved more potent than SJG-136 and all three compounds showed good activity against mycobacteria. There was little or no variation in susceptibility to each compound among isolates from any one species; this is exemplified in Table 1, which summarizes MIC data for the 38 MRSA isolates. Disc diffusion assays using the antibiotics listed in the Materials and methods section provided no evidence of cross-resistance between the three PBD dimers and other antibiotics (data not shown).


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Table 1. MIC values (mg/L) of PBD dimers against various bacterial species

 
Bactericidal activity of PBD dimers

The capacity of ELB-21 to kill EMRSA-16 and VRE was determined. As can be seen from Figure 2, ELB-21 exhibited potent bactericidal activity against logarithmic phase EMRSA-16 cells. Stationary phase cells were also efficiently killed by ELB-21 and the curves obtained were similar to those for logarithmic phase cells (data not shown). The re-growth that occurred after 24 h was not due to the emergence of resistant genotypes. Logarithmic phase VRE cells were highly susceptible to killing by ELB-21, with a five-log reduction in viability after 4 h incubation at the MIC. Incubation of EMRSA-16 with ELB-21 at the MIC for 4 h resulted in a marked degree of morphological heterogeneity and drug-induced lysis (Figure 3), suggesting that ELB-21 inhibits the transcription of a multiplicity of key genes.



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Figure 2. In vitro killing kinetics of ELB-21 against (a) logarithmic phase EMRSA-16 and (b) a logarithmic phase VRE isolate. The MICs for EMRSA-16 and VRE were 0.015 and 0.06 mg/L, respectively. Open squares, control; filled squares, MIC; filled triangles, twice the MIC; open circles, four times the MIC. Values shown are the averages of duplicate experiments and error bars indicate the range of values.

 


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Figure 3. Sections of glutaraldehyde-fixed EMRSA-16 stained with uranyl acetate and Reynold's lead citrate. (a) Untreated cells and (b) cells incubated with ELB-21 at the MIC for 4 h.

 
PAE

In vitro PAEs for EMRSA-16 and VRE were determined against ELB-21 over a range of concentrations. At the MIC, the PAEs for the two strains were 1.5 and 3.5 h, respectively. The PAE for VRE could not be determined at concentrations above the MIC due to rapid bacterial killing at these concentrations. PAEs against MRSA-16 at twice and four times the MIC were 1.5 and 3.5 h, respectively.

DNA melting

Covalent cross-linking of the two strands of the DNA duplex will increase the temperature required to denature the complex;17 melting of the complex results in a large increase in absorbance that can be monitored spectrophotometrically. Addition of 20 µM of either DRG-16 or ELB-21 to genomic EMRSA-16 DNA had a profound effect on the Tm: DRG-16 raised the Tm by, typically, 28.07°C (from 66.70 to 94.77°C) and ELB-21 by 30.81°C (error ±0.1°C).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The large majority of currently useful antibacterial agents have been derived from natural products produced by microorganisms forced to compete for nutrients in hostile habitats such as soil. The majority of targets for clinically useful natural and semi-synthetic antibiotics are enzymes or structural proteins involved in essential pathways of macromolecule synthesis and most of these have been extensively exploited for antibiotic development. Naturally occurring antibacterial agents such as distamycin, anthramycin and chromomycin that inhibit gene transcription by binding selectively to specific sequences of duplex DNA have been known for many years18 but it is only recently that efforts have been made to develop such agents as antibacterial drugs. Even though the simplest DNA binding antibiotics show selectivity for various sequence motifs,18 they are generally cytotoxic and appear unattractive as candidates for the treatment of infection in humans. However, the increasing prevalence of multidrug resistance, associated, in particular, with nosocomial pathogens has created circumstances under which such molecules may have therapeutic utility.

PBD dimers are attracting interest as potential therapeutics for the treatment of various cancers due to their capacity to recognize and cross-link DNA in a sequence-selective manner. In particular, the PBD dimer SJG-136, which produces interstrand cross-links at embedded Pu-GATC-Py target sites within duplex DNA,8,9 is currently in Phase I clinical development.19 This compound, comprised of two PBD units tethered through a three-carbon diether linkage (Figure 1), possesses potent bactericidal activity against the five species of Gram-positive bacteria used in this study (Table 1) and, in particular, all MRSA and VRE isolates proved to be highly susceptible. Searching the EMRSA-16 genome20 using the Artemis genome viewer (www.sanger.ac.uk/Software/Artemis) indicated that the preferred SJG-136 binding sequence AGATCT8,9 occurs 162 times within coding sequences, including pseudo-genes, and 109 times within non-coding regions of the EMRSA-16 genome; cross-linking at these sites is highly likely to account for its observed in vitro potency.

Loadman and coworkers have shown that after intraperitoneal administration of SJG-136 to NCR/Nu mice at the MTD (0.2 mg/kg), SJG-136 shows a favourable pharmacokinetic profile:19 high peak plasma concentrations of Cmax = 336 nM at 30 min after dosing were observed. A calculated terminal t1/2 of 0.98 h and AUC of 0.34 µM·h resulted in a clearance rate of 17.7 mL/min per kg. Furthermore, the dimer appeared to bind only moderately to plasma proteins. These data indicate that a therapeutic index for BPD dimers as antibacterial agents is achievable, at least in the case of SJG-136, as plasma drug concentrations are more than 10-fold higher than those required for antibacterial efficacy in vitro. These authors define the term ‘maximum tolerated dose’ that we used in the context of this study.

The PBD units within DRG-16 and ELB-21 are tethered through a five-carbon diether linker, rather than three as is the case for SJG-136 (Figure 1), and both are thought to recognize and bind to Pu-GAATC-Py sequences;8 these compounds, particularly ELB-21, are more potent than SJG-136 (Table 1). In contrast to distamycin-based polyheterocyclic antibiotics that, like vancomycin, are bactericidal against staphylococci but bacteriostatic against enterococci,4 PBD dimers are bactericidal against both MRSA and VRE with a significant PAE. Modelling of the interstrand DNA minor-groove cross-linked adducts formed with PBD dimers indicates that there is little or no disruption of DNA secondary structure including base-mispairing and that induced distortion effects are absent.10 As these compounds are well accommodated within the minor groove such that little of either PBD unit remains exposed beyond the periphery of the host DNA duplex, the complexes are resistant to repair enzymes that are reliant on tracing distortion or helical perturbation in DNA. Both halves of the dimer behave in an essentially identical fashion without disruption of DNA integrity, and the flexible regions linking the two subunits make favourable non-bonding contacts with the floor and walls of the minor groove to enhance overall binding.10 It is likely that the failure of repair surveillance mechanisms to detect sites of cross-linking accounts in large part for the bactericidal nature of these molecules as interstrand cross-links, if not repaired, will interfere with the process of DNA replication and result in cell death.

In conclusion, the preliminary data presented here indicate that these compounds exert their antibacterial effects by cross-linking duplex DNA at selected sites on the bacterial genome and these observations are currently being extended using a variety of techniques to quantify the extent and selective nature of the cross-linking process. The compounds appear to cross the bacterial cytoplasmic membrane efficiently but are unable to traverse the outer membrane of Gram-negative bacteria. The molecular weights of these relatively hydrophilic compounds fall within the 500–600 Da range and it is not therefore surprising that they are excluded from passage through the protein channels of this barrier.21 There may, however, be scope for synergy with compounds that disrupt the integrity of the outer membrane.

The basis of the increase in antibacterial potency of PBD dimers resulting from extension of the linker region is at present unclear and we are investigating whether these changes are accompanied by increases in cytotoxicity or genotoxicity. The potency of this new class of agents against Gram-positive bacteria is high and there appears to be an opportunity for the design of sequence-selective dimers that have a high affinity for motifs within the DNA of key Gram-positive pathogens.


    Acknowledgements
 
We thank Tony Reszka for valuable help with DNA melting experiments and Spirogen Ltd. for financial support. P. W. T. thanks the EPSRC for a QUOTA studentship award.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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6. Thurston DE. Advances in the study of pyrrolo[2,1-c][1,4]-benzodiazepine antitumour antibiotics. In: Waring MJ, Neidle S, eds. Molecular Aspects of Anticancer Drug–DNA Interactions. London: Macmillan, 1993; 54–88.

7. Puvvada MS, Forrow SA, Hartley JA et al. Inhibition of bacteriophage T7 RNA polymerase in vitro transcription by DNA-binding pyrrolo[2,1-c][1,4]-benzodiazepines. Biochemistry 1997; 36: 2478–84.[CrossRef][ISI][Medline]

8. Smellie M, Bose DS, Thompson AS et al. Sequence-selective recognition of duplex DNA through covalent interstrand cross-linking: kinetic and molecular modelling studies with pyrrolobenzodiazepine dimers. Biochemistry 2003; 42: 8232–9.[CrossRef][ISI][Medline]

9. Martin C, Ellis T, McGurk CJ et al. Sequence-selective interaction of the minor-groove interstrand cross-linking agent SJG-136 with naked and cellular DNA: footprinting and enzyme inhibition studies. Biochemistry 2005; 44: 4135–47.[CrossRef][ISI][Medline]

10. Gregson SJ, Howard PW, Hartley JA et al. Design, synthesis, and evaluation of a novel pyrrolobenzodiazepine DNA-interactive agent with highly efficient cross-linking ability and potent cytotoxicity. J Med Chem 2001; 44: 737–48.[CrossRef][ISI][Medline]

11. Gregson SJ, Howard PW, Gullick DR et al. Linker length modulates DNA cross-linking reactivity and cytotoxic potency of C8/C8' ether-linked C2-exo-unsaturated pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers. J Med Chem 2004; 47: 1161–74.[CrossRef][ISI][Medline]

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16. Jones GB, Davey CL, Jenkins TC et al. The non-covalent interaction of pyrrolo[2,1-c][1,4]benzodiazepine-5,11-diones with DNA. Anti-Cancer Drug Des 1990; 5: 249–64.[ISI][Medline]

17. James PL, Le Strat L, Ellervik U et al. Effects of a hairpin polyamide on DNA melting: comparison with distamycin and Hoechst 33258. Biophys Chem 2004; 111: 205–12.[CrossRef][ISI][Medline]

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