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Supplementary Figures 1-6, Table 1 from G-Quadruplexes Induce Apoptosis in Tumor Cells

Several G-rich oligodeoxynucleotides (ODNs), which are capable of forming G-quadruplexes, have been shown to exhibit antiproliferative activity against tumor cell lines and antitumor activity in nude mice carrying prostate and breast tumor xenografts. However, the molecular basis for their antitumor activity remains unclear. In the current study, we showed that a variety of telomeric G-tail oligodeoxynucleotides (TG-ODNs) exhibited antiproliferative activity against many tumor cells in culture. Systematic mutational analysis of the TG-ODNs suggests that the antiproliferative activity depends on the G-quadruplex conformation of these TG-ODNs. TG-ODNs were also shown to induce poly(ADP-ribose) polymerase-1 cleavage, phosphatidylserine flipping, and caspase activation, indicative of induction of apoptosis. TG-ODN–induced apoptosis was largely ataxia telangiectasia mutated (ATM) dependent. Furthermore, TG-ODN–induced apoptosis was inhibited by the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125. Indeed, TG-ODNs were shown to activate the JNK pathway in an ATM-dependent manner as evidenced by elevated phosphorylation of JNK and c-Jun. Interestingly, a number of G-quadruplex ODNs (GQ-ODN) derived from nontelomeric sequences also induced ATM/JNK-dependent apoptosis, suggesting a possible common mechanism of tumor cell killing by GQ-ODNs. (Cancer Res 2006; 66(24): 11808-16)

MreB is a cytoskeleton protein present in rod-shaped bacteria that is both essential for bacterial cell division and highly conserved. Because most Gram (-) bacteria require MreB for cell division, chromosome segregation, cell wall morphogenesis, and cell polarity, it is an attractive target for antibacterial drug discovery. As MreB modulation is not associated with the activity of antibiotics in clinical use, acquired resistance to MreB inhibitors is also unlikely. Compounds, such as A22 and CBR-4830, are known to disrupt MreB function by inhibition of ATPase activity. However, the toxicity of these compounds has hindered efforts to assess the in vivo efficacy of these MreB inhibitors. The present study further examines the structure-activity of analogs related to CBR-4830 as it relates to relative antibiotic activity and improved drug properties. These data reveal that certain analogs have enhanced antibiotic activity. In addition, we evaluated several representative analogs (9, 10, 14, 26, and 31) for their abilities to target purified E. coli MreB (EcMreB) and inhibit its ATPase activity. Except for 14, all these analogs were more potent than CBR-4830 as inhibitors of the ATPase activity of EcMreB with corresponding IC50 values ranging from 6.3 ± 2.0 to 29.4 ± 8.8 uM.

Addressing the growing problem of antibiotic resistance requires the development of new drugs with novel antibacterial targets. FtsZ has been identified as an appealing new target for antibacterial agents. Here, we describe the structure-guided design of a new fluorescent probe (BOFP) in which a BODIPY fluorophore has been conjugated to an oxazole-benzamide FtsZ inhibitor. Crystallographic studies have enabled us to identify the optimal position for tethering the fluorophore that facilitates the high-affinity FtsZ binding of BOFP. Fluorescence anisotropy studies demonstrate that BOFP binds the FtsZ proteins from the Gram-positive pathogens Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae with Kd values of 0.6–4.6 µM. Significantly, BOFP binds the FtsZ proteins from the Gram-negative pathogens Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii with an even higher affinity (Kd = 0.2–0.8 µM). Fluorescence microscopy studies reveal that BOFP can effectively label FtsZ in all the above Gram-positive and Gram-negative pathogens. In addition, BOFP is effective at monitoring the impact of non-fluorescent inhibitors on FtsZ localization in these target pathogens. Viewed as a whole, our results highlight the utility of BOFP as a powerful tool for identifying new broad-spectrum FtsZ inhibitors and understanding their mechanisms of action.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen of acute clinical importance. Combination treatment with an FtsZ inhibitor potentiates the activity of penicillin binding protein (PBP)-targeting β-lactam antibiotics against MRSA. To explore the mechanism underlying this synergistic behavior, we examined the impact of treatment with the FtsZ inhibitor TXA707 on the spatial localization of the five PBP proteins expressed in MRSA. In the absence of drug treatment, PBP1, PBP2, PBP3, and PBP4 colocalize with FtsZ at the septum, contributing to new cell wall formation. By contrast, PBP2a localizes to distinct foci along the cell periphery. Upon treatment with TXA707, septum formation becomes disrupted and FtsZ relocalizes away from mid-cell. PBP1 and PBP3 remain significantly colocalized with FtsZ, while PBP2, PBP4, and PBP2a localize away from FtsZ to specific sites along the periphery of the enlarged cells. We also examined the impact on PBP2a and PBP2 localization of treatment with β-lactam antibiotic oxacillin alone and in synergistic combination with TXA707. Significantly, PBP2a localizes to the septum in approximately 15% of the oxacillin-treated cells, a behavior that likely contributes to the β-lactam resistance of MRSA. Combination treatment with TXA707 causes both PBP2a and PBP2 to localize in malformed septal-like structures. Our collective results suggest that PBP2, PBP4, and PBP2a may function collaboratively in peripheral cell wall repair and maintenance in response to FtsZ inhibition by TXA707. Cotreatment with oxacillin, appears to reduce the availability of PBP2a to assist in this repair, thereby rendering the MRSA cells more susceptible to the β-lactam. IMPORTANCE MRSA is a multidrug-resistant bacterial pathogen of acute clinical importance, infecting many thousands of individuals globally each year. The essential cell division protein FtsZ has been identified as an appealing target for the development of new drugs to combat MRSA infections. Through synergistic actions, FtsZ-targeting agents can sensitize MRSA to antibiotics like the β-lactams that would otherwise be ineffective. This study provides key insights into the mechanism underlying this synergistic behavior as well as MRSA resistance to β-lactam drugs. The results of this work will help guide the identification and optimization of combination drug regimens that can effectively treat MRSA infections and reduce the potential for future resistance.

Several studies that have identified agents that potentiate the antimicrobial activity of antibiotics, but there are limited insights into their structure-activity relationships (SAR). The SAR associated with select N-alkylaryl amide derivatives of ornithine was performed to establish those structural features that were associated with potentiation of the antimicrobial activity of clarithromycin against E. coli ATCC 25922. The data indicate that the N-propyl derivative was slightly more active in reducing the effective MIC of clarithromycin against E. coli ATCC 25922. In addition, the S-enantiomer of compound 9 was somewhat more potent than the R-enantiomer in potentiating clarithromycin activity. No significant enhancement in potentiation activity was observed with the conversion of these secondary amides to their N-methyl tertiary amides. Formation of the N-methyl or N,N-dimethyl derivatives of the primary amine of 9 was associated with the loss of potentiation activity. Conversion of this primary amine to a guanidine was also not associated with an increase in potentiation activity. Among the isomeric diamino pentamides, 15 potentiated the antibacterial activity of clarithromycin to the greatest extent. In addition to these amide derivatives, the desoxy derivatives 16 and 18 were the more potent potentiators within this triamine series. The relative location of the primary amines, as indicated by the relative differences in the potentiation observed with 16 compared to 14, appears to be a critical factor in determining potentiation activity. Cell-based membrane permeabilization and efflux inhibition studies in E. coli ATCC 25922 suggest that the potentiation of clarithromycin activity by 16 reflects its ability to inhibit efflux pump activity and to a lesser extent its actions as a permeabilizer of the outer leaflet of the outer cell membrane.

Combination therapy of bacterial infections with synergistic drug partners offers distinct advantages over monotherapy. Among these advantages are (i) a reduction of the drug dose required for efficacy, (ii) a reduced potential for drug-induced toxicity, and (iii) a reduced potential for the emergence of resistance. Here, we describe the synergistic actions of the third-generation oral cephalosporin cefdinir and TXA709, a new, FtsZ-targeting prodrug that we have developed with improved pharmacokinetics and enhanced in vivo efficacy against methicillin-resistant Staphylococcus aureus (MRSA) relative to earlier agents. We show that the active product of TXA709 (TXA707) acts synergistically with cefdinir in vitro against clinical isolates of MRSA, vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA), and linezolid-resistant S. aureus (LRSA). In addition, relative to TXA707 alone, the combination of TXA707 and cefdinir significantly reduces or eliminates the detectable emergence of resistance. We also demonstrate synergy in vivo with oral administration of the prodrug TXA709 and cefdinir in mouse models of both systemic and tissue (thigh) infections with MRSA. This synergy reduces the dose of TXA709 required for efficacy 3-fold. Viewed as a whole, our results highlight the potential of TXA709 and cefdinir as a promising combination for the treatment of drug-resistant staphylococcal infections.

The clinical development of FtsZ-targeting benzamide compounds like PC190723 has been limited by poor drug-like and pharmacokinetic properties. Development of prodrugs of PC190723 (e.g., TXY541) resulted in enhanced pharmaceutical properties, which, in turn, led to improved intravenous efficacy as well as the first demonstration of oral efficacy in vivo against both methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA). Despite being efficacious in vivo , TXY541 still suffered from suboptimal pharmacokinetics and the requirement of high efficacious doses. We describe here the design of a new prodrug (TXA709) in which the Cl group on the pyridyl ring has been replaced with a CF 3 functionality that is resistant to metabolic attack. As a result of this enhanced metabolic stability, the product of the TXA709 prodrug (TXA707) is associated with improved pharmacokinetic properties (a 6.5-fold-longer half-life and a 3-fold-greater oral bioavailability) and superior in vivo antistaphylococcal efficacy relative to PC190723. We validate FtsZ as the antibacterial target of TXA707 and demonstrate that the compound retains potent bactericidal activity against S. aureus strains resistant to the current standard-of-care drugs vancomycin, daptomycin, and linezolid. These collective properties, coupled with minimal observed toxicity to mammalian cells, establish the prodrug TXA709 as an antistaphylococcal agent worthy of clinical development.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen that poses a significant risk to global health today. We have developed a promising new FtsZ-targeting agent (TXA707) with potent activity against MRSA isolates resistant to current standard-of-care antibiotics. We present here results that demonstrate differing extents of synergy between TXA707 and a broad range of β-lactam antibiotics (including six cephalosporins, two penicillins, and two carbapenems) against MRSA. To explore whether there is a correlation between the extent of synergy and the preferential antibacterial target of each β-lactam, we determined the binding affinities of the β-lactam antibiotics for each of the four native penicillin-binding proteins (PBPs) of S. aureus using a fluorescence anisotropy competition assay. A comparison of the resulting PBP binding affinities with our corresponding synergy results reveals that β-lactams with a high affinity for PBP2 afford the greatest degree of synergy with TXA707 against MRSA. In addition, we present fluorescence and electron microscopy studies that suggest a potential mechanism underlying the synergy between TXA707 and the β-lactam antibiotics. In this connection, our microscopy results show a disruption of septum formation in TXA707-treated MRSA cells, with a concomitant mislocalization of the PBPs from midcell to nonproductive peripheral sites. Viewed as a whole, our results indicate that PBP2-targeting β-lactam antibiotics are optimal synergistic partners with FtsZ-targeting agents for use in combination therapy of MRSA infections.

In the effort to combat antibiotic resistance, inhibitors of the essential bacterial protein FtsZ have emerged as a promising new class of compounds with clinical potential. One such FtsZ inhibitor (TXA707) is associated with potent activity against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) that are resistant to current standard-of-care antibiotics. However, mutations in S. aureus FtsZ (SaFtsZ) that confer resistance to TXA707 have been observed, with mutations in the Gly196 and Gly193 residues being among the most prevalent. Here, we describe structural studies of an FtsZ inhibitor, TXA6101, which retains activity against MRSA isolates that express either G196S or G193D mutant FtsZ. We present the crystal structures of TXA6101 in complex with both wild-type SaFtsZ and G196S mutant SaFtsZ, as well the crystal structure of TXA707 in complex with wild-type SaFtsZ. Comparison of the three structures reveals a molecular basis for the differential targeting abilities of TXA6101 and TXA707. The greater structural flexibility of TXA6101 relative to TXA707 enables TXA6101 to avoid steric clashes with Ser196 and Asp193. Our structures also demonstrate that the binding of TXA6101 induces previously unobserved conformational rearrangements of SaFtsZ residues in the binding pocket. In aggregate, the structures reported in this work reveal key factors for overcoming drug resistance mutations in SaFtsZ and offer a structural basis for the design of FtsZ inhibitors with enhanced antibacterial potency and reduced susceptibility to mutational resistance.

Infections caused by Gram-negative bacterial pathogens are often difficult to treat, with the emergence of multidrug-resistant strains further restricting clinical treatment options. As a result, there is an acute need for the development of new therapeutic agents active against Gram-negative bacteria. The bacterial protein FtsZ has recently been demonstrated to be a viable antibacterial target for treating infections caused by the Gram-positive bacteria Staphylococcus aureus in mouse model systems. Here, we investigate whether an FtsZ-directed prodrug (TXY436) that is effective against S. aureus can also target Gram-negative bacteria, such as Escherichia coli. We find that the conversion product of TXY436 (PC190723) can bind E. coli FtsZ and inhibit its polymerization/bundling in vitro. However, PC190723 is intrinsically inactive against wild-type E. coli, with this inactivity being derived from the actions of the efflux pump AcrAB. Mutations in E. coli AcrAB render the mutant bacteria susceptible to TXY436. We further show that chemical inhibition of AcrAB in E. coli, as well as its homologs in Klebsiella pneumoniae and Acinetobacter baumannii, confers all three Gram-negative pathogens with susceptibility to TXY436. We demonstrate that the activity of TXY436 against E. coli and K. pneumoniae is bactericidal in nature. Evidence for FtsZ-targeting and inhibition of cell division in Gram-negative bacteria by TXY436 is provided by the induction of a characteristic filamentous morphology when the efflux pump has been inhibited as well as by the lack of functional Z-rings upon TXY436 treatment.

The bacterial cell division protein FtsZ represents a novel antibiotic target that has yet to be exploited clinically. The benzamide PC190723 was among the first FtsZ-targeting compounds to exhibit in vivo efficacy in a murine infection model system. Despite its initial promise, the poor formulation properties of the compound have limited its potential for clinical development. We describe here the development of an N -Mannich base derivative of PC190723 with enhanced drug-like properties and oral in vivo efficacy. The N -Mannich base derivative (TXY436) is ∼100-fold more soluble than PC190723 in an acidic aqueous vehicle (10 mM citrate, pH 2.6) suitable for oral in vivo administration. At physiological pH (7.4), TXY436 acts as a prodrug, converting to PC190723 with a conversion half-life of 18.2 ± 1.6 min. Pharmacokinetic analysis following intravenous administration of TXY436 into mice yielded elimination half-lives of 0.26 and 0.96 h for the TXY436 prodrug and its PC190723 product, respectively. In addition, TXY436 was found to be orally bioavailable and associated with significant extravascular distribution. Using a mouse model of systemic infection with methicillin-sensitive Staphylococcus aureus or methicillin-resistant S. aureus , we show that TXY436 is efficacious in vivo upon oral administration. In contrast, the oral administration of PC190723 was not efficacious. Mammalian cytotoxicity studies of TXY436 using Vero cells revealed an absence of toxicity up to compound concentrations at least 64 times greater than those associated with antistaphylococcal activity. These collective properties make TXY436 a worthy candidate for further investigation as a clinically useful agent for the treatment of staphylococcal infections.

New antibiotics with novel mechanisms of action are urgently needed to overcome the growing bacterial resistance problem faced by clinicians today. PC190723 and related compounds represent a promising new class of antibacterial compounds that target the essential bacterial cell division protein FtsZ. While this family of compounds exhibits potent antistaphylococcal activity, they have poor activity against enterococci and streptococci. The studies described herein are aimed at investigating the molecular basis of the enterococcal and streptococcal resistance to this family of compounds. We show that the poor activity of the compounds against enterococci and streptococci correlates with a correspondingly weak impact of the compounds on the self-polymerization of the FtsZ proteins from those bacteria. In addition, computational and mutational studies identify two key FtsZ residues (E34 and R308) as being important determinants of enterococcal and streptococcal resistance to the PC190723-type class of compounds.

Bacterial cell division occurs in conjunction with the formation of a cytokinetic Z-ring structure comprised of FtsZ subunits. Agents that disrupt Z-ring formation have the potential, through this unique mechanism, to be effective against several of the newly emerging multidrug-resistant strains of infectious bacteria. Several 1-phenylbenzo[c]phenanthridines exhibit notable antibacterial activity. Based upon their structural similarity to these compounds, a distinct series of substituted 1,6-diphenylnaphthalenes were synthesized and evaluated for antibacterial activity against Staphylococcus aureus and Enterococcus faecalis. In addition, the effect of select 1,6-diphenylnaphthalenes on the polymerization dynamics of S. aureus FtsZ and mammalian β-tubulin was also assessed. The presence of a basic functional group or a quaternary ammonium substituent on the 6-phenylnaphthalene was required for significant antibacterial activity. Diphenylnaphthalene derivatives that were active as antibiotics, did exert a pronounced effect on bacterial FtsZ polymerization and do not appear to cross-react with mammalian tubulin to any significant degree.

Bacterial cell division occurs in conjunction with the formation of a cytokinetic Z-ring structure comprised of FtsZ subunits. Agents that can disrupt Z-ring formation have the potential, through this unique mechanism, to be effective against several of the newly emerging multi-drug resistant strains of infectious bacteria. 1- and 12-Aryl substituted benzo[c]phenanthridines have been identified as antibacterial agents that could exert their activity by disruption of Z-ring formation. Substituted 4- and 5-amino-1-phenylnaphthalenes represent substructures within the pharmacophore of these benzo[c]phenanthridines. Several 4- and 5-substituted 1-phenylnaphthalenes were synthesized and evaluated for antibacterial activity against Staphylococcus aureus and Enterococcus faecalis. The impact of select compounds on the polymerization dynamics of S. aureus FtsZ was also assessed.

The emergence of multidrug-resistant bacteria has created an urgent need for antibiotics with a novel mechanism of action. The bacterial cell division protein FtsZ is an attractive target for the development of novel antibiotics. The benzo[c]phenanthridinium sanguinarine and the dibenzo[a,g]quinolizin-7-ium berberine are two structurally similar plant alkaloids that alter FtsZ function. The presence of a hydrophobic functionality at either the 1-position of 5-methylbenzo[c]phenanthridinium derivatives or the 2-position of dibenzo[a,g]quinolizin-7-ium derivatives is associated with significantly enhanced antibacterial activity. 3-Phenylisoquinoline represents a subunit within the ring-systems of both of these alkaloids. Several 3-phenylisoquinolines and 3-phenylisoquinolinium derivatives have been synthesized and evaluated for antibacterial activity against Staphylococcus aureus and Enterococcus faecalis, including multidrug-resistant strains of methicillin-resistant S. aureus (MRSA) and vancomycin-resistant E. faecalis (VRE). A number of derivatives were found to have activity against both MRSA and VRE. The binding of select compounds to S. aureus FtsZ (SaFtsZ) was demonstrated and characterized using fluorescence spectroscopy. In addition, the compounds were shown to act as stabilizers of SaFtsZ polymers and concomitant inhibitors of SaFtsZ GTPase activity. Toxicological assessment of select compounds revealed minimal cross-reaction mammalian β-tubulin as well as little or no human cytotoxicity.

G-quadruplex stabilizers such as telomestatin and HXDV bind with exquisite specificity to G-quadruplexes, but not to triplex, duplex, or single-stranded DNAs. Studies have suggested that the antiproliferative and possibly anti-tumor activities of these compounds are linked to their inhibitory effect on telomerase and/or telomere function. In the current studies, we show that HXDV, a synthetic analog of telomestatin, exhibits antiproliferative activity against both telomerase-positive and -negative cells and induces robust apoptosis within 16 h of treatment, suggesting a mode of action independent of telomerase. HXDV was also shown to inhibit cell cycle progression causing M-phase cell cycle arrest, as evidenced by accumulation of cells with 4 n DNA content, increased mitotic index, separated centrosomes, elevated histone H3 phosphorylation at Ser-10 (an M-phase marker), and defective chromosome alignment and spindle fiber assembly (revealed by time-lapse microscopy). The M-phase arrest caused by HXDV paralleled with reduction in the expression level of the major M-phase checkpoint regulator Aurora A. All these cellular effects appear to depend on the G-quadruplex binding activity of HXDV as its non-G-quadruplex binding analog, TXTLeu, is completely devoid of all these effects. In the aggregate, our results suggest that HXDV, which exhibits anti-proliferative and apoptotic activities, is also a novel M-phase blocker, with a mode of action dependent on its G-quadruplex binding activity.

Nonsense mutations promote premature translational termination and represent the underlying cause of a large number of human genetic diseases. The aminoglycoside antibiotic gentamicin has the ability to allow the mammalian ribosome to read past a false-stop signal and generate full-length functional proteins. However, severe toxic side effects along with the reduced suppression efficiency at subtoxic doses limit the use of gentamicin for suppression therapy. We describe here the first systematic development of the novel aminoglycoside 2 (NB54) exhibiting superior in vitro readthrough efficiency to that of gentamicin in seven different DNA fragments derived from mutant genes carrying nonsense mutations representing the genetic diseases Usher syndrome, cystic fibrosis, Duchenne muscular dystrophy, and Hurler syndrome. Comparative acute lethal toxicity in mice, cell toxicity, and the assessment of hair cell toxicity in cochlear explants further indicated that 2 exhibits far lower toxicity than that of gentamicin.

Oxazole-containing macrocycles, which include the natural product telomestatin, represent a promising class of anticancer agents that target G-quadruplex DNA. Two synthetic hexaoxazole-containing macrocyclic compounds (HXDV and HXLV-AC) have been characterized with regard to their cytotoxic activities versus human cancer cells, as well as the mode, thermodynamics, and specificity with which they bind to the intramolecular (3+1) G-quadruplex structural motif formed in the presence of K+ ions by human telomeric DNA. Both compounds exhibit cytotoxic activities versus human lymphoblast (RPMI 8402) and oral carcinoma (KB3-1) cells, with associated IC50 values ranging from 0.4 to 0.9microM. The compounds bind solely to the quadruplex nucleic acid form, but not to the duplex or triplex form. Binding to the quadruplex is associated with a stoichiometry of two ligand molecules per DNA molecule, with one ligand molecule binding to each end of the host quadruplex via a nonintercalative "terminal capping" mode of interaction. For both compounds, quadruplex binding is primarily entropy driven, while also being associated with a negative change in heat capacity. These thermodynamic properties reflect contributions from favorable ligand-induced alterations in the loop configurational entropies of the quadruplex, but not from changes in net hydration. The stoichiometry and mode of binding revealed by our studies have profound implications with regard to the number of ligand molecules that can potentially bind the 3-overhang region of human telomeric DNA.

Macrocyclic hexaoxazoles having one or two lysinyl side chains in which the terminal nitrogen is either a primary amine, N,N-dimethylamine, or an acetamide have been synthesized. Sodium ion has been found to be beneficial to the macrocyclization step by acting as a template around which the linear polyoxazole can organize. Each of the targeted compounds selectivity stabilizes G-quadruplex versus duplex DNA. Compounds with one valine and one lysine residue display the best combination of G-quadruplex stabilizing ability with no detectable stabilization of duplex DNA.

International audience; The lack of absolute prokaryotic selectivity of natural antibiotics is widespread and is a significant clinical problem. The use of this disadvantage of aminoglycoside antibiotics for the possible treatment of human genetic diseases is extremely challenging. Here, we have used a combination of biochemical and structural analysis to compare and contrast the molecular mechanisms of action and the structure-activity relationships of a new synthetic aminoglycoside, NB33, and a structurally similar natural aminoglycoside apramycin. The data presented herein demonstrate the general molecular principles that determine the decreased selectivity of apramycin for the prokaryotic decoding site, and the increased selectivity of NB33 for the eukaryotic decoding site. These results are therefore extremely beneficial for further research on both the design of new aminoglycoside-based antibiotics with diminished deleterious effects on humans, as well as the design of new aminoglycoside-based structures that selectively target the eukaryotic ribosome.

2-Deoxystreptamine (2-DOS) aminoglycosides exert their antibiotic actions by binding to the A site of the 16S rRNA and interfering with bacterial protein synthesis. However, the molecular forces that govern the antitranslational activities of aminoglycosides are poorly understood. Here, we describe studies aimed at elucidating these molecular forces. In this connection, we compare the bactericidal, antitranslational, and rRNA binding properties of the 4,5-disubstituted 2-DOS aminoglycoside neomycin (Neo) and a conformationally restricted analog of Neo (CR-Neo) in which the 2′-nitrogen atom is covalently conjugated to the 5′′-carbon atom. The bactericidal potency of Neo exceeds that of CR-Neo, with this enhanced antibacterial activity reflecting a correspondingly enhanced antitranslational potency. Time-resolved fluorescence anisotropy studies suggest that the enhanced antitranslational potency of Neo relative to that of CR-Neo is due to a greater extent of drug-induced reduction in the mobilities of the nucleotides at positions 1492 and 1493 of the rRNA A site. Buffer- and salt-dependent binding studies, coupled with high-resolution structural information, point to electrostatic contacts between the 2′-amino functionality of Neo and the host rRNA as being an important modulator of 1492 and 1493 base mobilities and therefore antitranslational activities.

This unit describes the application of calorimetry to characterize the thermodynamics of nucleic acids, specifically, the two major calorimetric methodologies that are currently employed: differential scanning (DSC) and isothermal titration calorimetry (ITC). DSC is used to study thermally induced order-disorder transitions in nucleic acids. A DSC instrument measures, as a function of temperature (T), the excess heat capacity (C(p)(ex)) of a nucleic acid solution relative to the same amount of buffer solution. From a single curve of C(p)(ex) versus T, one can derive the following information: the transition enthalpy (ΔH), entropy (ΔS), free energy (ΔG), and heat capacity (ΔCp); the state of the transition (two-state versus multistate); and the average size of the molecule that melts as a single thermodynamic entity (e.g., the duplex). ITC is used to study the hybridization of nucleic acid molecules at constant temperature. In an ITC experiment, small aliquots of a titrant nucleic acid solution (strand 1) are added to an analyte nucleic acid solution (strand 2), and the released heat is monitored. ITC yields the stoichiometry of the association reaction (n), the enthalpy of association (ΔH), the equilibrium association constant (K), and thus the free energy of association (ΔG). Once ΔH and ΔG are known, ΔS can also be derived. Repetition of the ITC experiment at a number of different temperatures yields the ΔCp for the association reaction from the temperature dependence of ΔH.

Steady-state and time-resolved fluorescence techniques have been used to characterize the energetics and dynamics associated with the interaction of an E. coli 16 S rRNA A-site model oligonucleotide and four aminoglycoside antibiotics that exhibit a broad range of antibacterial activity. The results of these characterizations suggest that aminoglycoside-induced reduction in the mobility of an adenine residue at position 1492 of the rRNA A-site is a more important determinant of antibacterial activity than drug affinity for the A-site. This observation is consistent with a recently proposed model for the mechanism of protein synthesis inhibition by aminoglycosides that invokes a drug-induced alteration in the conformational equilibrium of the rRNA A-site (centered around the conserved adenine residues at positions 1492 and 1493), which, in turn, promotes an enhanced interaction between the rRNA and the minihelix formed by the tRNA anticodon and the mRNA codon, even when the anticodon is noncognate. Regarded as a whole, the results reported here indicate that the rational design of antibiotics that target the 16 S rRNA A-site requires consideration of not only the structure and energetics of the drug-RNA complex but also the dynamics associated with that complex.

RNA degradation via the ribonuclease H (RNase H) activity of human immunodeficiency virus type I (HIV-1) reverse transcriptase (RT) is a critical component of the reverse transcription process. In this connection, mutations of RT that inactivate RNase H activity result in noninfectious virus particles. Thus, interfering with the RNase H activity of RT represents a potential vehicle for the inhibition of HIV-1 replication. Here, we demonstrate an approach for inhibiting the RNase H activity of HIV-1 RT by targeting its RNA.DNA hybrid substrates. Specifically, we show that the binding of the 4,5-disubstituted 2-deoxystreptamine aminoglycosides, neomycin, paromomycin, and ribostamycin, to two different chimeric RNA-DNA duplexes, which mimic two distinct intermediates in the reverse transcription process, inhibits specific RT-mediated RNase H cleavage, with this inhibition being competitive in nature. UV melting and isothermal titration calorimetry studies reveal a correlation between the relative binding affinities of the three drugs for each of the chimeric RNA-DNA host duplexes and the relative extents to which the drugs inhibit RT-mediated RNase H cleavage of the duplexes. Significantly, this correlation also extends to the relative efficacies with which the drugs inhibit HIV-1 replication. In the aggregate, our results highlight a potential strategy for AIDS chemotherapy that should not be compromised by the unusual genetic diversity of HIV-1.

Spectroscopic and calorimetric techniques were employed to characterize and contrast the binding of the aminoglycoside paromomycin to three octamer nucleic acid duplexes of identical sequence but different strand composition (a DNA.RNA hybrid duplex and the corresponding DNA.DNA and RNA.RNA duplexes). In addition, the impact of paromomycin binding on both RNase H- and RNase A-mediated cleavage of the RNA strand in the DNA.RNA duplex was also determined. Our results reveal the following significant features: (i) Paromomycin binding enhances the thermal stabilities of the RNA.RNA and DNA.RNA duplexes to similar extents, with this thermal enhancement being substantially greater in magnitude than that of the DNA.DNA duplex. (ii) Paromomycin binding to the DNA.RNA hybrid duplex induces CD changes consistent with a shift from an A-like to a more canonical A-conformation. (iii) Paromomycin binding to all three octamer duplexes is linked to the uptake of a similar number of protons, with the magnitude of this number being dependent on pH. (iv) The affinity of paromomycin for the three host duplexes follows the hierarchy, RNA.RNA > DNA.RNA >> DNA.DNA. (v) The observed affinity of paromomycin for the RNA.RNA and DNA.RNA duplexes decreases with increasing pH. (vi) The binding of paromomycin to the DNA.RNA hybrid duplex inhibits both RNase H- and RNase A-mediated cleavage of the RNA strand. We discuss the implications of our combined results with regard to the specific targeting of DNA.RNA hybrid duplex domains and potential antiretroviral applications.

The antimicrobial agent pentamidine inhibits the self-splicing of the group I intron Ca.LSU from the transcripts of the 26S rRNA gene of Candida albicans, but the mechanism of pentamidine inhibition is not clear. We show that preincubation of the ribozyme with pentamidine enhances the inhibitory effect of the drug and alters the folding of the ribozyme in a pattern varying with drug concentration. Pentamidine at 25 microM prevents formation of the catalytically active F band conformation of the precursor RNA and alters the ribonuclease T1 cleavage pattern of Ca.LSU RNA. The effects on cleavage suggest that pentamidine mainly binds to specific sites in or near asymmetric loops of helices P2 and P2.1 on the ribozyme, as well as to the tetraloop of P9.2 and the loosely paired helix P9, resulting in an altered structure of helix P7, which contains the active site. Positively charged molecules antagonize pentamidine inhibition of catalysis and relieve the drug effect on ribozyme folding, suggesting that pentamidine binds to a magnesium binding site(s) of the ribozyme to exert its inhibitory effect.

We use spectroscopic and calorimetric techniques to characterize the binding of the aminoglycoside antibiotics neomycin, paromomycin, and ribostamycin to a RNA oligonucleotide that models the A-site of Escherichia coli 16S rRNA. Our results reveal the following significant features: (i) Aminoglycoside binding enhances the thermal stability of the A-site RNA duplex, with the extent of this thermal enhancement decreasing with increasing pH and/or Na(+) concentration. (ii) The RNA binding enthalpies of the aminoglycosides become more exothermic (favorable) with increasing pH, an observation consistent with binding-linked protonation of one or more drug amino groups. (iii) Isothermal titration calorimetry (ITC) studies conducted as a function of buffer reveal that aminoglycoside binding to the host RNA is linked to the uptake of protons, with the number of linked protons being dependent on pH. Specifically, increasing the pH results in a corresponding increase in the number of linked protons. (iv) ITC studies conducted at 25 and 37 degrees C reveal that aminoglycoside-RNA complexation is associated with a negative heat capacity change (Delta C(p)), the magnitude of which becomes greater with increasing pH. (v) The observed RNA binding affinities of the aminoglycosides decrease with increasing pH and/or Na(+) concentration. In addition, the thermodynamic forces underlying these RNA binding affinities also change as a function of pH. Specifically, with increasing pH, the enthalpic contribution to the observed RNA binding affinity increases, while the corresponding entropic contribution to binding decreases. (vi) The affinities of the aminoglycosides for the host RNA follow the hierarchy neomycinparomomycinribostamycin. The enhanced affinity of neomycin relative to either paromomycin or ribostamycin is primarily, if not entirely, enthalpic in origin. (vii) The salt dependencies of the RNA binding affinities of neomycin and paromomycin are consistent with at least three drug NH(3)(+) groups participating in electrostatic interactions with the host RNA. In the aggregate, our results reveal the impact of specific alterations in aminoglycoside structure on the thermodynamics of binding to an A-site model RNA oligonucleotide. Such systematic comparative studies are critical first steps toward establishing the thermodynamic database required for enhancing our understanding of the molecular forces that dictate and control aminoglycoside recognition of RNA.

Using the X-ray crystal structure of the human topoisomerase I (TOP1)-DNA cleavable complex, we have developed a general model for the ternary drug-DNA-TOP1 cleavable complex formed with camptothecin (CPT) and its analogues. This model has the drug intercalated between the -1 and +1 base pairs, with the E-ring pointing into the minor groove and the A-ring directed toward the major groove. The ternary complex is stabilized by an array of hydrogen bonding and hydrophobic interactions between the drug and both the enzyme and the DNA. Significantly, the proposed model is consistent with the current body of experimental mutation, cross-linking, and structure-activity data. In addition, the model reveals potential sites of interaction that can provide a rational basis for the design of next generation compounds as well as for de novo drug design.

5-Phenylterbenzimidazole (1) is active as a topoisomerase I poison (topo I) and is cytotoxic to human tumor cells. No cross-resistance was observed for 1 when it was evaluated against the camptothecin-resistant cell line, CPT-K5. Derivatives of 1 substituted at the 2"-position, however, did exhibit cross-resistance to this cell line. The basis for the resistance of this cell line towards CPT is that it possesses a mutant form of topo I. These results suggest that substituents at the 2"-position may be in proximity to the wild-type enzyme. Therefore, we hypothesized that terbenzimidazoles with 2"-substituents could be capable of interacting with the enzyme and thereby influence activity within this class of topo I poisons. 5-Phenylterbenzimidazoles with a hydroxy, hydroxymethyl, mercapto, amino, N-benzoylaminomethyl, chloro, and trifluoromethyl group at the 2"-position were synthesized. In addition, several 2"-ethyl-5-phenylterbenzimidazoles were prepared containing either a methoxy, hydroxy, amino, or N-acetylamino group at the 2-position of the ethyl side-chain. These 2"-substituted 5-phenylterbenzimidazoles were evaluated as topo I poisons and for cytotoxic activity. The presence of a strong electron-withdrawing group at the 2"-position, such as a chloro or trifluoromethyl group, did enhance both topo I poisoning activity and cytotoxicity. Studies on the relative DNA binding affinity of 1 to its 2"-amino and 2"-trifluoromethyl derivatives did exhibit a correlation with their relative differences in biological activity.

Crescent-shaped synthetic ligands containing aromatic amino acids have been designed for specific recognition of predetermined DNA sequences in the minor groove of DNA. Simple rules have been developed that relate the side-by-side pairings of Imidazole (Im) and Pyrrole (Py) amino acids to their predicted target DNA sequences. We report here thermodynamic characterization of the DNA-binding properties of the six-ring hairpin polyamide, ImImPy-gamma-PyPyPy-beta-Dp (where gamma = gamma-aminobutyric acid, beta = beta-alanine, and Dp = dimethylaminopropylamide). Our data reveal that, at 20 degrees C, this ligand binds with a relatively modest 1.8-fold preference for the designated match site, 5'-TGGTA-3', over the single base pair mismatch site, 5'-TGTTA-3'. By contrast, we find that the ligand exhibits a 102-fold greater affinity for its designated match site relative to the double base pair mismatch site, 5'-TATTA-3'. These results demonstrate that the energetic cost of binding to a double mismatch site is not necessarily equal to twice the energetic cost of binding to a single mismatch site. Our calorimetrically measured binding enthalpies and calculated entropy data at 20 degrees C reveal the ligand sequence specificity to be enthalpic in origin. We have compared the DNA-binding properties of ImImPy-gamma-PyPyPy-beta-Dp with the hairpin polyamide, ImPyPy-gamma-PyPyPy-beta-Dp (an Im --Py "mutant"). Our data reveal that both ligands exhibit high affinities for their designated match sites, consistent with the Dervan pairing rules. Our data also reveal that, relative to their corresponding single mismatch sites, ImImPy-gamma-PyPyPy-beta-Dp is less selective than ImPyPy-gamma-PyPyPy-beta-Dp for its designated match site. This result suggests, at least in this case, that enhanced binding affinity can be accompanied by some loss in sequence specificity. Such systematic comparative studies allow us to begin to establish the thermodynamic database required for the rational design of synthetic polyamides with predictable DNA-binding affinities and specificities.

The effect of DNA binding on poisoning of human DNA TOP1 has been studied using a pair of related anthracyclines which differ only by a nogalose sugar ring. We show that the nogalose sugar ring of nogalamycin, which binds to the minor groove of DNA, plays an important role in affecting topoisomerase-specific poisoning. Using purified mammalian topoisomerases, menogaril is shown to poison topoisomerase II but not topoisomerase I. By contrast, nogalamycin poisons topoisomerase I but not topoisomerase II. Consistent with the biochemical studies, CEM/VM-1 cells which express drug-resistant TOP2alpha are cross-resistant to menogaril but not nogalamycin. The mechanism by which nogalamycin poisons topoisomerase I has been studied by analyzing a major topoisomerase I-mediated DNA cleavage site induced by nogalamycin. This site is mapped to a sequence embedded in an AT-rich region with four scattered GC base pairs (bps) (at -10, -6, +2, and +12 positions). GC bps embedded in AT-rich regions are known to be essential for nogalamycin binding. Surprisingly, DNase I footprinting analysis of nogalamycin-DNA complexes has revealed a drug-free region from -2 to +9 encompassing the major cleavage site. Our results suggest that nogalamycin, in contrast to camptothecin, may stimulate TOP1 cleavage by binding to a site(s) distal to the site of cleavage.

Pyridyl polyoxazoles are 24-membered macrocyclic lactams comprised of a pyridine, four oxazoles and a phenyl ring. A derivative having a 2-(dimethylamino)ethyl chain attached to the 5-position of the phenyl ring was recently identified as a selective G-quadruplex stabilizer with excellent cytotoxic activity, and good in vivo anticancer activity against a human breast cancer xenograft in mice. Here we detail the synthesis of eight new dimethylamino-substituted pyridyl polyoxazoles in which the point of attachment to the macrocycle, as well as the distance between the amine and the macrocycle are varied. Each compound was evaluated for selective G-quadruplex stabilization and cytotoxic activity. The more active analogs have the amine either directly attached to, or separated from the phenyl ring by two methylene groups. There is a correlation between those macrocycles that are effective ligands for the stabilization of G-quadruplex DNA (DT(tran) 15.5-24.6 °C) and cytotoxicity as observed in the human tumor cell lines, RPMI 8402 (IC₅₀ 0.06-0.50 μM) and KB3-1 (IC₅₀ 0.03-0.07 μM). These are highly selective G-quadruplex stabilizers, which should prove especially useful for evaluating both in vitro and in vivo mechanism(s) of biological activity associated with G-quaqdruplex ligands.

The benzamide derivative PC190723 was among the first of a promising new class of FtsZ-directed antibacterial agents to be identified that exhibit potent antistaphylococcal activity. However, the compound is associated with poor drug-like properties. As part of an ongoing effort to develop FtsZ-targeting antibacterial agents with increased potential for clinical utility, we describe herein the pharmacodynamics, pharmacokinetics, in vivo antistaphylococcal efficacy, and mammalian cytotoxicity of TXY541, a novel 1-methylpiperidine-4-carboxamide prodrug of PC190723. TXY541 was found to be 143-times more soluble than PC190723 in an aqueous acidic vehicle (10mM citrate, pH 2.6) suitable for both oral and intravenous in vivo administration. In staphylococcal growth media, TXY541 converts to PC190723 with a half-life of approximately 8h. In 100% mouse serum, the TXY541-to-PC190723 conversion was much more rapid (with a half-life of approximately 3min), suggesting that the conversion of the prodrug in serum is predominantly enzyme-catalyzed. Pharmacokinetic analysis of both orally and intravenously administered TXY541 in mice yielded a half-life for the PC190723 conversion product of 0.56h and an oral bioavailability of 29.6%. Whether administered orally or intravenously, TXY541 was found to be efficacious in vivo in mouse models of systemic infection with both methicillin-sensitive and methicillin-resistant S. aureus. Toxicological assessment of TXY541 against mammalian cells revealed minimal detectable cytotoxicity. The results presented here highlight TXY541 as a potential therapeutic agent that warrants further pre-clinical development.

A series of macrocyclic biphenyl tetraoxazoles was synthesized. The latter stages of the synthetic approach allowed for the addition of varied N-protected α-amino acids, which were subsequently deprotected and condensed to provide the desired macrocycles. Improved yields could be realized in the macrocyclization step of their synthesis relative to other macrocyclic G-quadruplex stabilizers. These 24-membered macrocycles were evaluated for their ability to stabilize G-quadruplex DNA and for their relative cytotoxicity against human tumor cells. These biphenyl tetraoxazoles were not strong ligands for G-quadruplex DNA relative to other macrocyclic polyoxazoles. This reduced stabilizing potential did correlate with their comparatively lower cytotoxic activity as observed in the human tumor cell lines, RPMI 8402 and KB3-1. These studies provide useful insights into the conformational requirements for the development of selective and more potent G-quadruplex ligands.

Berenil is an antitrypanosomal agent that binds to nucleic acid duplexes. The generally accepted mode of berenil binding is via complexation into the minor groove of AT-rich domains of DNA double helices. We find that berenil can bind to RNA as well as DNA duplexes, while exhibiting properties characteristic of both intercalation as well as minor groove binding. More specifically, we use spectroscopic, calorimetric, and hydrodynamic techniques to characterize berenil binding to four DNA duplexes and to one RNA duplex. Our results reveal the following features: (i) Berenil binding to the poly[d(A-T)]2, poly(dA).poly(dT), poly[d(I-C)]2, poly[d(G-C)]2, and poly(rA).poly(rU) duplexes exhibits intercalative as well as minor groove binding characteristics. (ii) The apparent "site sizes" associated with berenil binding to these five duplexes range from 1 to 13 base pairs per bound berenil and depend, in part, on the host duplex. One of the site sizes common to all five duplexes is consistent with berenil binding to the minor groove. (iii) The apparent berenil binding affinity follows the hierarchy: poly(dA).poly(dT) > poly-[d(A-T)]2 approximately poly[d(I-C)]2 >> poly(rA).poly(rU) > poly[d(G-C)]2. (iv) Viscometric data reveal properties characteristic of a significant contribution from an intercalative mode of binding when berenil interacts with the poly[d(A-T)]2, poly[d(I-C)]2, poly[d(G-C)]2, and poly(rA).poly(rU) duplexes, while revealing an apparent nonintercalative mode when the drug binds to the poly(dA).poly(dT) duplex. (v) Berenil binding unwinds negative supercoils in the pBR322 plasmid, an observation consistent with an intercalative mode of binding to duplex DNA. (vi) Salt-dependent melting data suggest that both positively charged amidino groups of berenil participate in the complexation of the drug to the poly[d(I-C)]2, poly[d(A-T)]2, poly(dA).poly(dT), and poly(rA).poly(rU) duplexes, while also suggesting that the binding event is site-specific. In the aggregate, our results suggest that, in contrast to the conventional wisdom, berenil can exhibit intercalative as well as minor groove binding properties when it binds to both DNA and RNA duplexes, while also exhibiting a preference for DNA duplexes with unobstructed minor grooves. We comment on the potential correlation between drugs, such as berenil, that exhibit "mixed" binding motifs and those that express anticancer activity via inhibition of topoisomerase I activity.

It is becoming increasingly apparent that energetic as well as structural information is required to develop a complete appreciation of the critical interrelationships between structure, energetics, and biological function. Motivated by this recognition, we have reviewed in this article the current state of the thermodynamic databases associated with lesion-containing DNA duplexes and DNA quadruplexes, while highlighting important considerations concerning the methods used to obtain the requisite data

The prevalence of multidrug resistance among clinically significant bacterial pathogens underscores a critical need for the development of new classes of antibiotics with novel mechanisms of action. Here we describe the synthesis and evaluation of a guanidinomethyl biaryl compound {1-((4'-(tert-butyl)-[1,1'-biphenyl]-3-yl)methyl)guanidine} that targets the bacterial cell division protein FtsZ. In vitro studies with various bacterial FtsZ proteins reveal that the compound alters the dynamics of FtsZ self-polymerization via a stimulatory mechanism, while minimally impacting the polymerization of tubulin, the closest mammalian homologue of FtsZ. The FtsZ binding site of the compound is identified through a combination of computational and mutational approaches. The compound exhibits a broad spectrum of bactericidal activity, including activity against the multidrug-resistant pathogens methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE), while also exhibiting a minimal potential to induce resistance. Taken together, our results highlight the compound as a promising new FtsZ-targeting bactericidal agent.

Antibiotic resistance has prompted efforts to discover antibiotics with novel mechanisms of action. FtsZ is an essential protein for bacterial cell division, and has been viewed as an attractive target for the development of new antibiotics. Sanguinarine is a benzophenanthridine alkaloid that prevents cytokinesis in bacteria by inhibiting FtsZ self-assembly. In this study, a series of 5-methylbenzo[c]phenanthridinium derivatives were synthesized and evaluated for antibacterial activity against Staphylococcus aureus and Enterococcus faecalis. The data indicate that the presence of a 1- or 12-phenyl substituent on 2,3,8,9-tetramethoxy-5-methylbenzo[c]phenanthridinium chloride significantly enhances antibacterial activity relative to the parent compound or sanguinarine.

Berberine is a substituted dibenzo[a,g]quinolizin-7-ium derivative whose modest antibiotic activity is derived from its disruptive impact on the function of the essential bacterial cell division protein FtsZ. The present study reveals that the presence of a biphenyl substituent at either the 2- or 12-position of structurally-related dibenzo[a,g]quinolizin-7-ium derivatives significantly enhances antibacterial potency versus Staphylococcus aureus and Enterococcus faecalis. Studies with purified S. aureus FtsZ demonstrate that both 2- and 12-biphenyl dibenzo[a,g]quinolizin-7-ium derivatives act as enhancers of FtsZ self-polymerization.

The self-association and DNA-binding properties of two benzopyridoindole derivatives, 3-methoxy-7H-8-methyl-11-[(3'-aminopropyl)amino]benzo[e]pyrido[4,3-b]indole (BePI) and 3-methoxy-7-[((3'-diethylamino)propyl)amino]-10-methyl-11IH-benzo[g]pyrido[4,3-b]indole (BgPI), have been investigated by a variety of NMR, spectrophotometric, fluorescence, and hydrodynamic techniques. NMR studies indicate that both BePI and BgPI self-associate in solution, probably forming multimers (n-mers) in the process. BgPI self-associates with a 3-fold (at 80 o C) to 12-fold (at 27 o C) higher affinity than BePI. Self-association interactions do not interfere with those between either ligand and DNA at total ligand concentrations ≤10 -5 M

In an effort to probe the structure and stability of the CH + -C base pair in DNA, we have studied the conformation of the deoxydecanucleotide d(C 3 T 4 C 3 ) at pH 5.5, using 1 H-NMR and UV absorption spectroscopy. Lowering the pH of a solution containing d(C 3 T 4 C 3 ) is accompanied by the induction of six new imino proton NMR resonances in a region (15.415.8 ppm) downfield from where either T-H 3 or G-H1 Watson-Crick (W-C)-paired imino protons normally resonate

The synthesis of a series of 24-membered pyridine-containing polyoxazole macrocycles is described. Seventeen new macrocycles were evaluated for cytotoxic activity against RPMI 8402, KB-3, and KB-3 cell lines that overexpress the efflux transporters MDR1 (KBV-1) and BCRP (KBH5.0). Macrocycles in which the pyridyl-polyoxazole moiety is linked by a 1,3-bis(aminomethyl)phenyl group with a 5-(2-aminoethyl)-18, or a 5-(2-dimethylaminoethyl)-substituent 19, displayed the greatest cytotoxic potency. These compounds exhibit exquisite selectivity for stabilizing G-quadruplex DNA with no stabilization of duplex DNA or RNA. Compound 19 stabilizes quadruplex mRNA that encodes the cell-cycle checkpoint protein kinase Aurora A to a greater extent than the quadruplex DNA of a human telomeric sequence. These data may suggest a prominent role for G-quadruplex ligands interacting with mRNA being associated with the biological activity of macrocyclic polyoxazoles. Compound 19 has significant in vivo anticancer activity against a human breast cancer xenograft (MDA-MB-435) in athymic nude mice.