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PubMed1990

DNA-directed alkylating agents. 3. Structure-activity relationships for acridine-linked aniline mustards: consequences of varying the length of the linker chain.

Four series of acridine-linked aniline mustards have been prepared and evaluated for in vitro cytotoxicity, in vivo antitumor activity, and DNA cross-linking ability. The anilines were attached to the DNA-intercalating acridine chromophores by link groups (-O-, -CH2-, -S-, and -SO2-) of widely varying electronic properties, providing four series of widely differing mustard reactivity where the alkyl chain linking the acridine and mustard moieties was varied from two to five carbons. Relationships were sought between chain length and biological properties. Within each series, increasing the chain length did not alter the reactivity of the alkylating moiety but did appear to position it differently on the DNA, since cross-linking ability (measured by agarose gel assay) altered with chain length, being maximal with the C4 analogue. The in vivo antitumor activities of the compounds depended to some extent on the reactivity of the mustard, with the least reactive SO2 compounds being inactive. However, DNA-targeting did appear to allow the use of less reactive mustards, since the S-linked acridine mustards showed significant activity whereas the parent S-mustard did not. Within each active series, the most active compound was the C4 homologue, suggesting some relationship between activity and extent of DNA alkylation.

Acridines↗

PubMedDate not supplied

Identification of the major lesion from the reaction of an acridine-targeted aniline mustard with DNA as an adenine N1 adduct.

DNA adducts of two acridine-linked aniline half-mustards have been isolated and identified. The compound where the half-mustard is attached to the DNA-targeting acridine moiety by a short linker chain alkylates both double- and single-stranded DNA exclusively at guanine N7, as do the majority of known aromatic and aliphatic nitrogen mustards. The longer-chain analogue, also containing a more reactive half-mustard, shows a strikingly different pattern, alkylating double-stranded DNA to yield primarily (> 90%) the adenine N1 adduct, together with < 10% of the adenine N3 adduct and only trace amounts of the guanine N7 adduct. In the presence of MgCl2 (which is known not to inhibit the interaction of drugs at minor groove sites), the adenine N3 adduct is the major product. The latter compound is the first known aniline mustard (and apparently the first known alkylating agent of any type) to preferentially alkylate adenine at the N1 position in duplex DNA. These results are consistent with previous work [Prakash et al. (1990) Biochemistry 29, 9799-9807], which showed that the preferred site of DNA alkylation by the corresponding long-chain acridine-linked aniline bis-mustards in general was at major groove sites of adenines and identifies the major site of alkylation as adenine N1 and not N7. This selectivity for adenine N1 alkylation is suggested to result from a preference for the acridine mustard side chain of these compounds to project into the major groove following intercalation of the acridine, coupled with structural distortion of the DNA helix to make the N1 positions of adenines adjacent to the intercalation sites more accessible.

Acridines↗

PubMed2026

Pervasive phosphorylation by phage T7 kinase disarms bacterial defences.

Bacteria and bacteriophages are in a constant arms race to develop defence and anti-defence systems, respectively. Currently known phage-encoded anti-defence systems are specific to the activity of the targeted bacterial defence system. Here we identify a mechanism by which the T7 bacteriophage broadly counteracts bacterial defences using protein phosphorylation. Its kinase (T7K), which has been reported to redirect the function of a few host proteins1-5, is actually a hyperpromiscuous dual-specificity kinase that phosphorylates nearly all host and phage proteins during infection. The scale of phosphorylation vastly exceeds known phosphosites in Escherichia coli, has no sequence motif specificity and results in a higher proteome-wide phosphorylation density than mammalian cells with around 500 kinases. Stoichiometry analysis of phosphorylation sites revealed strong bias in T7K activity towards nucleic-acid-binding substrates mediated by its C-terminal DNA-binding domain. This highly stoichiometric phosphorylation enables the deactivation of DNA-targeting or DNA-containing bacterial defence systems. We provide mechanistic insights into how T7K weakens DNA-containing Retron-Eco9 through specific phosphorylation events, with single phosphomimetic mutations in key sites of the toxin abolishing defence. Moreover, by screening a large collection of E. coli strains, we provide evidence of broad anti-defence abilities of T7K in nature, as counteracted strains contain diverse bacterial defence systems. T7K homologues are found almost exclusively in phages, with hyperpromiscuous kinase activity probably being enabled by a divergent DFG-like motif in the catalytic centre.

Journal Article↗

PubMed1998

Human normal peripheral blood B-lymphocytes are deficient in DNA-dependent protein kinase activity due to the expression of a variant form of the Ku86 protein.

The heterodimeric Ku protein, which comprises a 86 kDa (Ku86) amd a 70 kDa (Ku70) subunits, is an abundant nuclear DNA-binding protein which binds in vitro to DNA termini without sequence specificity. Ku is the DNA-targeting component of the large catalytic sub-unit of the DNA-dependent protein kinase complex (DNA-PK[CS]), that plays a critical role in mammalian double-strand break repair and lymphoid V(D)J recombination. By using electrophoretic mobility shift assays, we demonstrated that in addition to the major Ku x DNA complex usually detected in cell line extracts, a second complex with faster electrophoretic mobility was observed in normal peripheral blood lymphocytes (PBL) extracts. The presence of this faster migrating complex was restricted to B cells among the circulating lymphocyte population. Western blot analysis revealed that B cells express a variant form of the Ku86 protein with an apparent molecular weight of 69 kDa, and not the 86 kDa- full-length protein. Although the heterodimer Ku70/variant-Ku86 binds to DNA-ends, this altered form of the Ku heterodimer has a decreased ability to recruit the catalytic component of the complex, DNA-PK(CS), which contributes to an absence of detectable DNA-PK activity in B cells. These data provide a molecular basis for the increased sensitivity of B cells to ionizing radiation and identify a new mechanism of regulation of DNA-PK activity that operates in vivo.

Antigens, Nuclear↗

PubMed1995

Targeted disruption of the beta 3-adrenergic receptor gene.

beta 3-Adrenergic receptors (beta 3-ARs) are expressed predominantly in white and brown adipose tissue, and beta 3-selective agonists are potential anti-obesity drugs. However, the role of beta 3-ARs in normal physiology is unknown. To address this issue, homologous recombination was used to generate mice that lack beta 3-ARs. This was accomplished by direct injection of a DNA-targeting construct into mouse zygotes. Twenty-three transgenic mice were generated, of which two had targeted disruption of the beta 3-AR gene. Mice that were homozygous for the disrupted allele had undetectable levels of intact beta 3-AR mRNA, as assessed by RNase protection assay and Northern blotting, and lacked functional beta 3-ARs, as demonstrated by complete loss of beta 3-agonist (CL 316,243)-induced stimulation of adenylate cyclase activity and lipolysis. beta 3-AR-deficient mice had modestly increased fat stores (females more than males), indicating that beta 3-ARs play a role in regulating energy balance. Importantly, beta 1 but not beta 2-AR mRNA levels up-regulated in white and brown adipose tissue of beta 3-AR-deficient mice (brown more than white), strongly implying that beta 3-ARs mediate physiologically relevant signaling under normal conditions and that "cross-talk" exists between beta 3-ARs and beta 1-AR gene expression. Finally, acute treatment of normal mice with CL 316,243 increased serum levels of free fatty acids (FFAs) (3.2-fold) and insulin (140-fold), increased energy expenditure (2-fold), and reduced food intake (by 45%). These effects were completely absent in beta 3-AR-deficient mice, proving that the actions of CL are mediated exclusively by beta 3-ARs. beta 3-AR-deficient mice should be useful as a means to a better understanding of the physiology and pharmacology of beta 3-ARs.

Adenylyl Cyclases↗

PubMed1995

Interaction with DNA of photoactive viologens based on the 6-(2- pyridinium)phenanthridinium structure.

A new type of DNA-interacting violgens derived from the N,N'-dialkyl 6-(2-pyridinium)-phenanthridinium structure (in which dialkyl is -CH2CH2-,-CH2CH2CH2-, or (-CH3)2) have been synthesized. Electronic spectroscopy, steady-state and time-resolved fluorescence, cyclic voltammetry, binding isotherms, viscosity titrations, and molecular modeling techniques were employed to characterize the structural, photophysical and redox properties of the novel drugs as well as the corresponding drug-DNA complexes. The viologens display significant visible absorption (up to ca. 490 nm), and a rather intense luminescence (phi cm from 0.06 to 0.20 at 491-565 nm wavelength maxima) which is efficiently quenched by DNA. The calculated redox potentials of these drugs in their singlet excited state (+2.1 V vs. SHE) predict a large driving force for a photoelectron transfer reaction from the nucleobases to the drugs. Photochemical measurements of the viologens in the presence of mononucleotides, nucleosides, and deoxyribose indicate that the observed fluorescence quenching occurs indeed by electron transfer from the DNA bases rather than the sugar phosphate backbone. Large association constants to double helical DNA (in the order of 10(5) M-1) have been evaluated from the absorbance-based binding isotherms. Viscosimetry supports intercalation of the drugs into the DNA helix. Computer simulations (molecular mechanics of d(CGCGCG)2-drug complexes) confirm the intercalative nature of the binding and provide finer details about the geometry of the different viologen-DNA complexes. Molecular modeling has also revealed a stereoselective interaction of the enantiomeric drug conformers with the chiral DNA helix. A DNA-targeted drug design of future generations of these ligands in order to improve and/or modulate their photochemical, redox, and nucleic acid binding properties appears to be possible by a careful selection of the N,N'-dialkylating chain and/or the substituents on the azaheterocyclic moieties.

Base Sequence↗

PubMed1988

DNA cross-linking and cytotoxicity of the alkylating cyanomorpholino derivative of doxorubicin in multidrug-resistant cells.

The cyanomorpholino derivative of doxorubicin (MRA-CN) is an anthracycline that is extremely potent and non-cross-resistant with doxorubicin (DOX) in multidrug-resistant cells. MRA-CN binds to and cross-links DNA and thus has been proposed to act as a targeted alkylating agent. In our study, the number of DNA interstrand and DNA-protein cross-links produced by MRA-CN was identical in multidrug-resistant Dx5 and parental MES-SA cells, as shown by alkaline elution analysis. The amount of cross-linking was directly proportional to drug concentration at concentrations from 10(-11) to 10(-7) M MRA-CN. Extensive DNA cross-linking was evident within 30 minutes of drug exposure. After 1 hour of drug exposure, the number of DNA cross-links increased for 90 minutes, reached a plateau, and then began to decrease after 120 minutes. Loss of cell viability was also observed as early as 3 hours after exposure to MRA-CN. The finding of the same number of DNA cross-links in MES-SA and Dx5 cells indicates that similar amounts of MRA-CN are likely to enter the nuclei of multidrug-resistant and sensitive cells. Other anthracyclines have major differences in nuclear distribution in sensitive and resistant cells. Several factors may contribute to the non-cross-resistance of MRA-CN in multidrug-resistant cells. (a) The lipophilicity of MRA-CN facilitates cell entry. (b) The substitution and loss of basicity at the amino nitrogen may reduce the affinity of the drug for the P-glycoprotein efflux pump, compared with that of DOX. (c) The detoxification function of P-glycoprotein may be less effective for drugs that produce rapid and irreversible cell damage, such as the DNA-targeted alkylation caused by MRA-CN.

Alkylating Agents↗

PubMed1992

DNA intercalation and photosensitization by cationic meso substituted porphyrins.

Several cationic porphyrins are known to bind to DNA by intercalative and outside binding modes. This study identifies the cis and trans isomers of bis(N-methyl-4-phridiniumyl)diphenyl porphyrin as DNA intercalators based on evidence from a DNA topoisomerase I assay. Moreover, both isomers are shown to be potent photosensitizers of DNA, inducing multiple S1 nuclease sensitive breaks in the phosphodiester backbone. Porphyrin-induced photodamage in DNA was also shown to be quantitatively dependent upon ionic strength and to inhibit the action of restriction endonucleases. The results indicate that these porphyrins can be useful probes of DNA structure and have potential as DNA-targeted photosensitizers.

Cations↗

PubMed1997

Protein-DNA interactions in the human beta-globin locus control region hypersensitive site-2 as revealed by four nitrogen mustards.

Four nitrogen mustards have been used in this study to examine protein-DNA interactions in intact human cells, specifically at the locus control region hypersensitive site-2 (LCR HS-2) of the human beta-globin locus. Three of these nitrogen mustards are DNA-targeted by attachment of an acridine or amsacrine intercalating chromophore, while the fourth (chlorambucil) is a non-targeted mustard. The ligation-mediated PCR technique was used to determine the sites of damage at base pair resolution on DNA sequencing gels. A densitometric comparison was made between DNA damaged in intact erythroid K562 cells and in purified DNA. The intensity of DNA damage sites in the LCR HS-2 were found to differ significantly between intact K562 cells and purified DNA. At the NF-E2/AP-1 motif, pronounced damage protection was observed in DNA derived from drug treated cells. The nuclear factor- erythroid 2 (NF-E2) protein factor is thought to bind at this NF-E2/AP-1 motif in K562 cells. Other sites of protection and enhancement that corresponded to known transcription factor binding sites were also detected. These nitrogen mustards are therefore very effective compounds for detection of transcription factor binding to DNA in intact cells and are superior to other commonly used agents. The sequence selectivity of the compounds was determined using plasmid DNA and compared to that found in intact cells. The acridine-based nitrogen mustard had a preference for forming adducts at guanine bases, while the two amsacrine-based nitrogen mustards and chlorambucil formed adducts at both guanine and adenine bases.

Aminacrine↗

PubMed2024

An essential and highly selective protein import pathway encoded by nucleus-forming phage.

UNLABELLED: Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here we identify two components of this novel protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA, that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together our results allow us to propose a multistep model for the Protein Import Chimallivirus (PIC) pathway, where proteins are targeted to PicA by amino acids on their surface, and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely-related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts. SIGNIFICANCE STATEMENT: The phage nucleus is an enclosed replication compartment built by Chimalliviridae phages that, similar to the eukaryotic nucleus, separates transcription from translation and selectively imports certain proteins. This allows the phage to concentrate proteins required for DNA replication and transcription while excluding DNA-targeting host defense proteins. However, the mechanism of selective trafficking into the phage nucleus is currently unknown. Here we determine the region of a phage nuclear protein that targets it for nuclear import and identify a conserved, essential nuclear shell-associated protein that plays a key role in this process. This work provides the first mechanistic model of selective import into the phage nucleus.

Preprint↗

PubMed1998

DNA end-independent activation of DNA-PK mediated via association with the DNA-binding protein C1D.

DNA-dependent protein kinase (DNA-PK), which is involved in DNA double-strand break repair and V(D)J recombination, is comprised of a DNA-targeting component termed Ku and an approximately 465-kD catalytic subunit, DNA-PKcs. Although DNA-PK phosphorylates proteins in the presence of DSBs or other discontinuities in the DNA double helix in vitro, the possibility exists that it is also activated in other circumstances via its association with additional proteins. Here, through use of the yeast two-hybrid screen, we discover that the recently identified high affinity DNA binding protein C1D interacts with the putative leucine zipper region of DNA-PKcs. Furthermore, we show that C1D can interact with DNA-PK in mammalian cells and that C1D is a very effective DNA-PK substrate in vitro. Finally, we establish that C1D directs the activation of DNA-PK in a manner that does not require DNA termini. Therefore, these studies provide a function for C1D and suggest novel mechanisms for DNA-PK activation in vivo.

3T3 Cells↗

PubMed1992

Anticellular and antitumor activity of duocarmycins, novel antitumor antibiotics.

The anticellular and antitumor activities of novel antitumor antibiotics, duocarmycins (DUMs), were examined against human and murine tumor cells. DUMs consist of five compounds, A, B1, B2, C1 and C2, which possess a pharmacophore similar to that of CC-1065, a previously isolated antibiotic. Among them, DUMA exhibited ultrapotent growth-inhibitory activity with an IC50 value of 6 pM against human uterine cervix carcinoma HeLa S3 cells. DUMA and DUMB1 also inhibited the growth of adriamycin (ADM)-resistant lines of human nasopharynx carcinoma KB cells and breast carcinoma MCF-7 cells as well as their sensitive lines. DUMs inhibited the growth of s.c.-inoculated murine tumors such as B16 melanoma, sarcoma 180, M5076 sarcoma and colon 26. DUMs were also significantly effective in increasing the lifespan of i.p.-inoculated B16 melanoma-bearing mice, although their effect was marginal against other i.p.-inoculated tumors. As a whole, DUMB1 exhibited superior activity to the other four compounds. DUMB1 rapidly inhibited the incorporation of [3H]-TdR into macromolecules of HeLa S3 cells as compared with that of [3H]UR or [3H]leucine. DNA strand breaks were detected in DUMB1-treated HeLa S3 cells by agarose gel electrophoresis with a contour-clamped homogeneous electric field apparatus. These results indicate that DUMs possess interesting biological activities as DNA-targeting antitumor antibiotics.

Animals↗

PubMed1998

Cloning and biochemical characterization of TAF-172, a human homolog of yeast Mot1.

The TATA binding protein (TBP) is a central component of the eukaryotic transcriptional machinery and is the target of positive and negative transcriptional regulators. Here we describe the cloning and biochemical characterization of an abundant human TBP-associated factor (TAF-172) which is homologous to the yeast Mot1 protein and a member of the larger Snf2/Swi2 family of DNA-targeted ATPases. Like Mot1, TAF-172 binds to the conserved core of TBP and uses the energy of ATP hydrolysis to dissociate TBP from DNA (ADI activity). Interestingly, ATP also causes TAF-172 to dissociate from TBP, which has not been previously observed with Mot1. Unlike Mot1, TAF-172 requires both TBP and DNA for maximal (approximately 100-fold) ATPase activation. TAF-172 inhibits TBP-driven RNA polymerase II and III transcription but does not appear to affect transcription driven by TBP-TAF complexes. As it does with Mot1, TFIIA reverses TAF-172-mediated repression of TBP. Together, these findings suggest that human TAF-172 is the functional homolog of yeast Mot1 and uses the energy of ATP hydrolysis to remove TBP (but apparently not TBP-TAF complexes) from DNA.

Adenosine Triphosphatases↗

PubMed1996

The DNA-dependent protein kinase, DNA-PK: 10 years and no ends in sight.

The phenomenon of DNA-dependent protein phosphorylation was first described little over 10 years ago. Since then a DNA-dependent protein kinase, DNA-PK, has been purified from human cells and many of its biochemical properties have been characterized. DNA-PK is composed of a large catalytic subunit, DNA-PKcs, and a DNA-targeting protein, Ku. When assembled on a suitable DNA molecule, the DNA-PK holoenzyme acts as a serine/threonine protein kinase that in vitro phosphorylates many DNA binding and non-binding proteins and transcription factors. Recent genetic studies point strongly to functions in DNA double-strand break repair and V(D)J recombination. In addition, biochemical studies suggest a role in the regulation of transcription. Here we discuss, from a historical perspective, the events leading up to our current understanding of the function of DNA-PK, including recent results from our own studies suggesting the involvement of DNA-PK in apoptosis and in viral infection of human cells.

Animals↗

PubMed1996

Novel aromatic urea derivatives with DNA-binding ability.

Several aromatic urea derivatives were designed and synthesized as DNA-targeting agents. N,N'-Dimethyl-N,N'-bis[(4-amidylphenyl)aminocarbonyl]-2,6-di aminopyridine (1) and 1,3-bis[5-(glycylamino)pyrid-2-yl]urea (3) showed remarkable DNA-binding abilities as determined by ultrafiltration assay using calf thymus DNA, their potencies being equal to and half that of netropsin, respectively. Compound 1 inhibited the proliferation of both L1210 cells and KB cells with similar IC50 values to netropsin.

Animals↗

PubMedDate not supplied

Phage T4 DNA [N6-adenine] methyltransferase: kinetic studies using oligonucleotides containing native or modified recognition sites.

The DNA-[N6-adenine] methyltransferase of T4 phage (T4 Dam MTase) catalyzes methyl group transfer from S-adenosyl-L-methionine (AdoMet) to the N6-position of adenine in the palindromic sequence, GATC. We have investigated the effect of eliminating different structural components of the recognition site on the ability of a substrate to be bound and methylated by T4 Dam. For this purpose, steady state binding (by gel shift assays) and kinetic parameters of methylation (using the methyl donor, [3H-CH3]-AdoMet, at 25 degrees C) were studied using various synthetic duplex oligonucleotides containing some defect in the DNA-target site; e.g., the absence of an internucleotide phosphate or a nucleotide(s) within the recognition site, or a single stranded region. The salient results are summarized as follows: (1) Addition of T4 Dam to a complete reaction mixture (with a 20-mer duplex as substrate) resulted in a 'burst' of 3H-methylated product, followed by a constant rate of product formation that reflected establishment of steady-state conditions. This suggests that the rate-limiting step is release of product methylated DNA from the enzyme [and not the transfer of the methyl group]. (2) A number of the defects in duplex structure had only a weak influence on the binding and Km values, but strongly reduced the kcat. At the same time, several poorly bound duplexes retained good substrate characteristics, especially duplexes having uninterrupted GAT-sequences in both strands. Whereas having only one half of the recognition site element intact was sufficient for stable complex formation, the catalytic turnover process had a strict requirement for an uninterrupted GAT-sequence on both strands. (3) There was no correlation between Km and binding capability; the apparent Kd for some duplexes was 5-70 times higher than Km. This indicates that the T4 Dam methylation reaction can not be explained by a simple Michaelian scheme.

Bacteriophage T4↗

PubMed1994

Free-radical repair by a novel perthiol: reversible hydrogen transfer and perthiyl radical formation.

2-(3-Aminopropyl-amino) ethaneperthiol (RSSH, the perthiol analogue of the thiol radioprotector, WR-1065) reacts with the alpha-hydroxy alkyl radical (CH3)2C.OH by donating a hydrogen atom as indicated by the characterization of perthiyl radicals (RSS.; lambda max approximately 374 nm, epsilon 374 approximately 1680 +/- 20 dm3 mol-1 cm-1) by pulse radiolysis. The perthiyl radical abstracts a hydrogen from the alcohol to establish a reversible hydrogen-transfer equilibrium. This equilibrium lies predominantly on the side of radical repair since the rate constants for the forward and reverse reactions at pH 4 are: kappa(RSSH+(CH3)2C.OH) = (2.4 +/- 0.1) x 10(9) dm3 mol-1 s-1 and kappa(RSS.+(CH3)2CHOH) = (3.8 +/- 0.3) x 10(3) dm3 mol-1 s-1 respectively. The pKa (RSSH<-->RSS(-)+H+) = 6.2 +/- 0.1 was determined from the pH dependence of the rate of perthiol repair. Identical experiments have been performed with WR-1065 allowing a direct comparison of free-radical repair reactivity to be made with the parthiol analogue. At pH approximately 7.4 the reactivities of the thiol and perthiol were similar, both repairing the alcohol radical with a rate constant of approximately (2.4 +/- 0.1) x 10(8) dm3 mol-1 s-1. However, at pH 5 whilst the hydrogen-donation rate of the thiol was 15-20% higher than at pH 7.4, the perthiol reactivity was over an order of magnitude higher. The thermodynamic driving force for the observed enhanced free-radical repair reactivity of RSSH compared to RSH is attributed to the resonance stabilization energy of 8.8 kJ mol-1 within the RSS. radical. These results indicate a possible application of RSSH/RSS- as DNA-targeted antioxidants or chemoprotectors.

Free Radicals↗

PubMed1992

5-Nitro-4-(N,N-dimethylaminopropylamino)quinoline (5-nitraquine), a new DNA-affinic hypoxic cell radiosensitizer and bioreductive agent: comparison with nitracrine.

Targeting of electron-affinic radiosensitizers to DNA via noncovalent binding (e.g., intercalation) may offer the potential for increasing sensitizing efficiency. However, it has been suggested that high-affinity DNA binding may compromise sensitization by restricting the mobility of sensitizers along the DNA, and by decreasing rates of extravascular diffusion in tumors. The weak DNA intercalator nitracrine (1-NC) is a more efficient radiosensitizer than related nitroacridines with higher DNA-binding affinities (Roberts et al., Radiat. Res. 123, 153-164, 1990). The present study investigates whether electron-affinic agents of even lower DNA-binding affinity may be superior to nitroacridines. The quinoline analog of 1-NC, 5-nitraquine (5-NO), was shown to have an intrinsic association constant for calf thymus DNA in 20 mM phosphate buffer which was 12-fold lower than that of 1-NC. 5-Nitraquine was not accumulated as efficiently as 1-NC by AA8 cells, but, despite a similar one-electron reduction potential, was 2- to 3-fold more potent than 1-NC as a hypoxia-selective radiosensitizer in vitro when compared on the basis of average intracellular concentration. Thus the radiosensitizing potency of 5-NQ appears not to be compromised by its low DNA-binding affinity. The cytotoxic mechanisms of 5-NQ and 1-NC appear to be similar (hypoxia-selective formation of DNA monoadducts), but 5-NQ is 1200-fold less potent than 1-NC as a cytotoxin. Despite this advantage, 5-NQ was not active in vivo as a radiosensitizer in SCCVII tumors. This lack of activity appears to be due to its relatively high toxicity in vivo (intraperitoneal LD50 of 105 mumol kg-1 in C3H/HeN mice), high one-electron reduction potential (-286 mV), and rapid metabolism to the corresponding amine in mice. The in vitro therapeutic index (hypoxic radiosensitizing potency/aerobic cytotoxic potency) of this weak DNA binder was lower than that of the non-DNA targeted radiosensitizer misonidazole, suggesting that DNA targeting enhances cytotoxicity more than radiosensitization. Development of useful DNA-targeted radiosensitizers may require the exploitation of DNA binding modes different from those of the nitroacridines and nitroquinolines.

Aminoquinolines↗