An unusually cold active nitroreductase for prodrug activations.

A set of PCR primers based on the genome sequence were used to clone a gene encoding a hypothetical nitroreductases (named as Ssap-NtrB) from uropathogenic staphylococcus, Staphylococcus saprophyticus strain ATCC 15305, an oxygen insensitive flavoenzyme. Activity studies of the translation product revealed that the nitroreductase catalyses two electron reduction of a nitroaromatic drug of nitrofurazone (NFZ), cancer prodrugs of CB1954 and SN23862 at optimum temperature of 20 °C together with retaining its maximum activity considerably at 3 °C. The required electrons for such reduction could be supplied by either NADH or NADPH with a small preference for the latter. The gene was engineered for heterologous expression in Escherichia coli, and conditions were found in which the enzyme was produced in a mostly soluble form. The recombinant enzyme was purified to homogeneity and physical, spectral and catalytical properties were determined. The findings lead us to propose that Ssap-NtrB represents a novel nitro reductase with an unusual cold active property, which has not been described previously for prodrug activating enzymes of nitroreductases.

Significant differences in biological parameters between prodrugs cleavable by carboxypeptidase G2 that generate 3,5-difluoro-phenol and -aniline nitrogen mustards in gene-directed enzyme prodrug therapy systems.

Nine new nitrogen mustard compounds derived from 2,6-difluoro-4-hydroxy- (3a-e) and 2,6-difluoro-4-amino- (4a-d) aniline were synthesized as potential prodrugs. They were designed to be activated to their corresponding 3,5-difluorophenol and -aniline (4)-nitrogen mustards by the enzyme carboxypeptidase G2 (CPG2) in gene-directed enzyme prodrug therapy (GDEPT) models. The compounds were tested for cytotoxicity in the MDA MB-361 breast adenocarcinoma. The cell line was engineered to express stably either CPG2 tethered to the cell surface stCPG2-(Q)3 or beta-galactosidase (beta-Gal) as control. The cytotoxicity differentials were calculated between CPG 2-expressing and -nonexpressing cells and yielded different results for the two series of prodrugs despite their structural similarities. While the phenol compounds are ineffective as prodrugs, their aniline counterparts exhibit outstanding activity in the tumor cell lines expressing CPG2. [3,5-Difluoro-4-[bis(2-chloroethyl)amino]phenyl]carbamoyl-l-glutamic acid gave a differential of >227 in MDA MB361 cells as compared with 19 exhibited by 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-l-glutamic acid, 1a, which has been in clinical trials.

2-Amino metabolites are key mediators of CB 1954 and SN 23862 bystander effects in nitroreductase GDEPT.

An important feature of gene-directed enzyme-prodrug therapy is that prodrug activation can provide diffusible cytotoxic metabolites capable of generating a local bystander effect in tumours. Activation of the aziridinyl dinitrobenzamide CB 1954 by E. coli nitroreductase (NTR) provides a bystander effect assumed to be due to the potently cytotoxic 4-hydroxylamine metabolite. We show that there are four cytotoxic extracellular metabolites of CB 1954 in cultures of NTR-expressing tumour cells (the 2- and 4-hydroxylamines and their corresponding amines). The 4-hydroxylamine is the most cytotoxic in DNA crosslink repair defective cells, but the 2-amino derivative (CB 10-236) is of similar potency to the 4-hydroxylamine in human tumour cell lines. Importantly, CB 10-236 has much superior diffusion properties to the 4-hydroxylamine in multicellular layers grown from the SiHa human cervical carcinoma cell line. These results suggest that the 2-amine, not the 4-hydroxylamine, is the major bystander metabolite when CB 1954 is activated by NTR in tumours. The corresponding dinitrobenzamide nitrogen mustard SN 23862 is reduced by NTR to form a single extracellular metabolite (also the 2-amine), which has superior cytotoxic potency and diffusion properties to the CB 1954 metabolites. These results are consistent with the reported high bystander efficiency of SN 23862 as an NTR prodrug in multicellular layers and tumour xenografts.

Studies on the nitroreductase prodrug-activating system. Crystal structures of complexes with the inhibitor dicoumarol and dinitrobenzamide prodrugs and of the enzyme active form.

The E. coli nitroreductase enzyme (NTR) has been widely used in suicide gene therapy (GDEPT and ADEPT) applications as a activating enzyme for nitroaromatic prodrugs of the dinitrobenzamide class. NTR has been previously shown to be a homodimeric enzyme with two active sites. We present here the crystal structures of the reduced form of NTR and its complexes with the inhibitor dicoumarol and three dinitrobenzamide prodrugs. Comparison of the structures of the oxidized and reduced forms of the native enzyme shows that the principal structural changes occur in the FMN cofactor and indicate that the enzyme itself is a relatively rigid structure that primarily provides a rigid structural framework on which hydride transfer occurs. The aziridinyldinitrobenzamide prodrug CB 1954 binds in nonidentical ways in both of the two active sites of the homodimeric enzyme, employing both hydrophobic and (in active site B) a direct H-bond contact to the side chain of Lys14. In active site A the 2-nitro group stacks above the FMN, and in active site B the 4-nitro group does, explaining why reduction of either nitro group is observed. In contrast, the larger mustard group of the dinitrobenzamide mustard compound SN 23862 forces the prodrug to bind at both active sites with only the 2-nitro group able to participate in hydride transfer from the FMN, explaining why only the 2-hydroxylamine reduction product is observed. In each site, the nitro groups of the prodrug make direct H-bond contacts with the enzyme; in active Site A the 2-nitro to Ser40 and the 4-nitro to Asn71, while in active Site B the 2-nitro contacts the main chain nitrogen of Thr41 and the 4-nitro group the Lys14 side chain. The related amide-substituted mustard SN 27217 binds in a broadly similar fashion, but with the larger amide group substituent able to reach and contact the side chain of Arg107, further restricting the prodrug conformations in the binding site. The inhibitor dicoumarol appears to bind primarily by pi-stacking interactions and hydrophobic contacts, with no conformational changes in the enzyme. One of the hydroxycoumarin subunits stacks above the plane of the FMN via pi-overlap with the isoalloxazine ring, penetrating deep into the groove, with the other less well-defined. These studies suggest guidelines for further prodrug design. Steric bulk (e.g., mustard rather than aziridine) on the ring can limit the possible binding orientations, and the reducible nitro group must be located para to the mustard. Substitution on the carboxamide side chain still allows the prodrugs to bind, but also limits their orientation in the binding site. Finally, modulating substrate specificity by alteration of the structure of the enzyme rather than the prodrug might usefully focus on modifying the Phe124 residue and those surrounding it.

Effect of nitroreduction on the alkylating reactivity and cytotoxicity of the 2,4-dinitrobenzamide-5-aziridine CB 1954 and the corresponding nitrogen mustard SN 23862: distinct mechanisms of bioreductive activation.

The dinitrobenzamide aziridine CB 1954 (1) and its nitrogen mustard analogue SN 23862 (6) are prodrugs that are activated by enzymatic nitroreduction in tumors. Bioactivation of 1 is considered to be due to reduction of its 4-nitro group to the hydroxylamine and subsequent formation of the N-acetoxy derivative; this acts as a reactive center, in concert with the aziridine moiety, to provide a bifunctional DNA cross-linking agent (Knox model). It is currently unclear whether bioactivation of 6 occurs by the same mechanism or results from the electronic effects of nitroreduction on reactivity of the nitrogen mustard moiety. To discriminate between these mechanisms, we have synthesized the hydroxylamine and amine derivatives of 1 and 6, plus related compounds, and determined their alkylating reactivities in aqueous solution, using LC/MS to identify reaction pathways. The relationships between substituent electronic effects, reactivity, and cytotoxicity were determined using the UV4 cell line, which is defective in nucleotide excision repair (thus avoiding differences in repair kinetics). Alkylating reactivity correlated with the electron-donating character of the ortho or para substituent in the case of the mustards, with a less marked electronic effect for the aziridines. Importantly, there was a highly significant linear relationship between cytotoxic potency and alkylating reactivity in both the aziridine and the mustard series, with the notable exception of 4, the 4-hydroxylamine of 1, which was 300-fold more toxic than predicted by this relationship. This demonstrates that the high potency of 4 does not result from activation of the aziridine ring, supporting the Knox model. The single-step bioactivation of 6, to amino or hydroxylamine metabolites with similar potency to 4, is a potential advantage in the use of dinitrobenzamide mustards as prodrugs for activation by nitroreductases.

A rationally designed genotoxin that selectively destroys estrogen receptor-positive breast cancer cells.

We describe a novel strategy to increase the selective toxicity of genotoxic compounds. The strategy involves the synthesis of bifunctional molecules capable of forming DNA adducts that have high affinity for specific proteins in target cells. It is proposed that the association of such proteins with damaged sites in DNA can compromise protein function and/or DNA repair resulting in increased toxicity. We describe the synthesis of a bifunctional compound consisting of an aniline mustard linked to the 7alpha position of estradiol. This novel compound can form covalent DNA adducts that have high affinity for the estrogen receptor. Breast cancer cells that express high levels of the estrogen receptor showed increased sensitivity to the cytotoxic effects of the new compound.

Pharmacokinetics and metabolism of the nitrogen mustard bioreductive drug 5.

PURPOSE: To characterise the pharmacokinetics and metabolism in mice of 5-[N,N-bis(2-chloroethyl)amino]-2,4-dinitrobenzamide (SN 23862), the lead compound of a new class of bioreductive drugs in which a nitrogen mustard is activated by nitroreduction. Comparison is made with the corresponding aziridine derivative CB 1954. METHODS: Male C3H/HeN mice, bearing s.c. KHT tumours, received 3H-labelled SN 23862 or CB 1954 i.v. at 200 micromol/kg. Plasma, urine and tumour samples were assayed for total radioactivity, and for parent compounds by HPLC. Metabolites were identified by 1H-NMR and mass spectrometry. Cytotoxicity of compounds against Chinese hamster AA8 cells was determined by growth inhibition assay. RESULTS: The plasma pharmacokinetics of SN 23862 and CB 1954 were similar, with half-lives of 1.1 and 1.2 h, respectively. SN 23862 provided tumour/plasma ratios and absolute tumour AUC values almost two times higher than CB 1954. Despite this, SN 23862 was more extensively metabolised than CB 1954, the major route being sequential oxidative dechloroethylation of the nitrogen mustard moiety to the relatively non-toxic half mustard and 5-amine. The inferred chloroacetaldehyde co-product was 260 times more potent than SN 23862. A tetrahydroquinoxaline metabolite resulting from reduction of the 4-nitro group followed by intramolecular alkylation was weakly cytotoxic, while the more cytotoxic 2-amino derivative of SN 23862 was detected in trace amounts. CB 1954 was metabolised by analogous pathways, but the 4- and 2-amine nitroreduction products were the major metabolites while oxidative dealkylation was minor. CONCLUSION: The lesser propensity for SN 23862 to undergo nitroreduction in the host, relative to CB 1954, argues that dinitrobenzamide mustards may be preferable to the corresponding aziridines as bioreductive prodrugs for cancer treatment. However, the toxicological significance of oxidative metabolism of the bis(2-chloroethyl)amine moiety needs to be addressed.

Sequence selectivity, cross-linking efficiency and cytotoxicity of DNA-targeted 4-anilinoquinoline aniline mustards.

We have investigated the sequence selectivity, DNA binding site characteristics, interstrand cross-linking ability and cytotoxicity of four 4-anilinoquinoline aniline mustards related to the AT-selective minor groove-binding bisquaternary ammonium heterocycles. The compounds studied include two full mustards that differ in alkylating power, a half mustard and a quaternary anilinoquinolinium bismustard. We have also compared their cytotoxicity with their precursor diols and their toxicity and cross-linking ability with the classical alkylating agents melphalan and chlorambucil. We find that the anilinoquinoline aniline mustards weakly and non-specifically alkylate guanines in the major groove and that they bind strongly to AT-rich sequences in the minor groove, where they alkylate both adenines and guanines at the N3 position. The most preferred sites are classical minor groove binder AT-tracts to which all four ligands bind equally well. The remaining sites are AT-rich, but include GC base pairs, to which the ligands bind with preferences depending on their structure. The full mustards alkylate at the 3' ends of the binding site in an orientation that depends on the spatial disposition of the purines within the two strands. Generally speaking guanines are found to be much less reactive than adenines. The anilinoquinoline aniline mustards are interstrand cross-linking agents that are 60- to 100-fold more effective than melphalan, with the quaternary compound being the most efficacious. However, the type of binding site at which the cross-links occur is not clear, since distamycin challenge fails to antagonize them fully. The full mustards are 20- to 50-fold more cytotoxic than their diol precursors, are more cytotoxic than the half mustard and are 20- to 30-fold more active than melphalan and chlorambucil. The quaternary ligand is the most potent. Given the evidence to hand, it appears that antitumour activity correlates with capacity to cause interstrand cross-links at classical or near-classical AT-minor groove binder sites, rather than with ability to discriminate between the subsets of potential anilinoquinoline aniline mustard binding sites.

Characterization of an antineoplastic glucuronide prodrug.

The specificity of tumor therapy may be improved by preferentially activating antineoplastic prodrugs at tumor cells pretargeted with antibody-enzyme conjugates. In this study, the conditions required for the efficient activation of p-hydroxyaniline mustard glucuronide (BHAMG) to p-hydroxyaniline mustard (pHAM) were investigated. pHAM induced cross-links in linearized double-stranded DNA at about 180-fold lower concentrations than BHAMG, indicating that the nucleophilicity of pHAM was decreased by the presence of a glucuronide group. The partition coefficient of BHAMG was about 1890 times lower than pHAM in an octanol-water two-phase system, suggesting that the reduced toxicity of BHAMG was due to both hindered diffusion across the lipid bilayer of cells and decreased reaction with nuclear DNA. BHAMG was significantly less toxic to BHK cells that expressed cytosolic Escherichia coli-derived beta-glucuronidase (betaG) compared with cells that were engineered to secrete betaG, demonstrating that extracellular localization of betaG was required for optimal activation of BHAMG. The extended retention of mAb RH1 on the surface of AS-30D cells was also consistent with extracellular activation of BHAMG. Taken together, our results indicate that the low toxicity of BHAMG was due to hindered cellular uptake and low alkylating activity. BHAMG must be enzymatically activated outside of tumor cells for maximum cytotoxicity, and non-internalizing antibodies are preferred for human tumor therapy by targeted antibody-enzyme activation of BHAMG.

Accelerated clearance of polyethylene glycol-modified proteins by anti-polyethylene glycol IgM.

Tumor therapy by the preferential activation of a prodrug at tumor cells targeted with an antibody-enzyme conjugate may allow improved treatment efficacy with reduced side effects. We examined antibody-mediated clearance of poly(ethylene glycol)-modified beta-glucuronidase (betaG-sPEG) as a method to reduce serum concentrations of enzyme and minimize systemic prodrug activation. Enzyme-linked immunosorbent assay and immunoblot analysis of two monoclonal antibodies generated by immunization of BALB/c mice with an antibody-betaG-sPEG conjugate showed that mAb 1E8 (IgG1) bound betaG and betaG-sPEG whereas mAb AGP3 (IgM) bound poly(ethylene glycol). Neither antibody affected the betaG activity. mAb 1E8 and AGP3 were modified with 36 and 208 galactose residues (1E8-36G and AGP3-208G) with retention of 72 and 48% antigen-binding activity, respectively, to target immune complexes to the asialoglycoprotein receptor on liver cells. mAb 1E8 and AGP3 cleared betaG-PEG from the circulation of mice as effectively as 1E8-36G and AGP3-208G, respectively. mAb AGP3, however, cleared betaG-sPEG more completely and rapidly than 1E8, reducing the serum concentration of betaG-sPEG by 38-fold in 8 h. AGP3 also reduced the concentration of an antibody-betaG-sPEG conjugate in blood by 280-fold in 2 h and 940-fold in 24 h. AGP3-mediated clearance did not produce obvious damage to liver, spleen, or kidney tissues. In addition, AGP3 clearance of betaG-sPEG before administration of BHAMG, a glucuronide prodrug of p-hydroxyaniline mustard, prevented toxicity associated with systemic activation of the prodrug based on mouse weight and blood cell numbers. AGP3 should be generally useful for accelerating the clearance of PEG-modified proteins as well as for improving the tumor/blood ratios of antibody-betaG-PEG conjugates for glucuronide prodrug therapy of cancer.

Bystander killing of tumour cells by antibody-targeted enzymatic activation of a glucuronide prodrug.

RHI-betaG-PEG, formed by linking poly(ethylene glycol)-modified beta-glucuronidase to Mab RH1, was employed to examine bystander killing of antigen-negative N1S1 rat hepatoma cells by activation of a glucuronide prodrug (BHAMG) of p-hydroxyaniline mustard (pHAM) at antigen-positive AS-30D rat hepatoma cells. Sequential treatment of cells with 10 microg ml(-1) RH1-betaG-PEG and 20 microM BHAMG was not toxic to N1S1 cells but killed 99% of AS-30D cells. Over 98% of N1S1 cells, however, were killed in mixed populations containing as few as 2% AS-30D cells after identical treatment, demonstrating an in vitro bystander effect. Subcutaneous injection of AS-30D and N1S1 cells in BALB/c nu/nu mice produced solid tumours containing both cells. Uptake of radiolabelled RH1-betaG-PEG in solid AS-30D and mixed AS-30D/N1S1 tumours was 11.6 and 9.3 times greater than a control antibody conjugate 120 h after i.v. injection. Intravenous treatment with RH1-betaG-PEG and BHAMG cured seven of seven nude mice bearing solid s.c. AS-30D tumours and significantly delayed, compared with control conjugate and prodrug treatment, the growth of mixed N1S1/AS-30D tumours with one cure, showing that targeted activation of BHAMG kills bystander tumour cells in vivo.

Cure of malignant ascites and generation of protective immunity by monoclonal antibody-targeted activation of a glucuronide prodrug in rats.

We examined the in vivo efficacy of targeting beta-glucuronidase (betaG) to activate a glucuronide prodrug (BHAMG) of p-hydroxyaniline mustard (pHAM) at hepatoma ascites in Sprague-Dawley rats. Injection i.p. of 500 microg RH1-betaG, a conjugate formed between recombinant betaG and monoclonal antibody RH1 with specificity for an antigen expressed on AS-30D rat hepatoma cells, into rats bearing AS-30D ascites resulted in the accumulation of 54 microg conjugate per 10(9) tumor cells after 2 hr. Ascites fluid and serum contained 0.53 and 0 microg/ml, respectively, RH1-betaG 2 hr after injection of the conjugate. Conjugate binding to AS-30D cells was heterogeneous and non-saturated, as determined by flow cytometry. BHAMG was less toxic than pHAM to SD rats based on measures of animal mortality, weight loss and hematological toxicity. Treatment of rats bearing established hepatoma ascites with 500 microg RH1-betaG followed 2 hr later with a single i.p. injection of 30 mg/kg BHAMG or 3 i.p. injections of 10 mg/kg BHAMG 2, 3 and 4 hr later resulted in the cure of 6/8 and 8/8 animals, respectively. Treatment with BHAMG or pHAM alone did not produce cures, whereas treatment with a control antibody-betaG conjugate and BHAMG produced significantly greater hematological toxicity compared to treatment with RH1-betaG and BHAMG. All cured rats were completely protected from rechallenge with 2 x 10(7) AS-30D cells, indicating that successful treatment of animals induced protective immunity.

Kinetics of the conjugation of aniline mustards with glutathione and thiosulfate.

The rates of the non-enzymatic conjugation of the substituted aniline mustards, melphalan, chlorambucil and p-(N,N-bis(2-chloroethyl))toluidine with glutathione and thiosulfate were determined using nuclear magnetic resonance spectroscopy. Using this method, the disappearance of drug and the formation of both the mono-thioether and bis-thioether conjugates can be monitored directly. For glutathione conjugation, the rate constants for the formation of the first and second aziridinium intermediates were similar. With thiosulfate conjugation, the rate constant for the formation of the first aziridinium intermediate is greater than the rate constant for the formation of the second aziridinium. This demonstrates that the type of nucleophile has a significant influence on the overall alkylating activity of these bifunctional mustards. The bisthioether adduct formed from the reaction between p-(N,N-bis([2-13C]-2-chloroethyl))toluidine and glutathione and thiosulfate can be identified and scrambling of the 13C label in the product provides strong evidence that the alkylation must occur through an aziridinium intermediate.

New mustard prodrugs for antibody-directed enzyme prodrug therapy: alternatives to the amide link.

Antibody-directed enzyme prodrug therapy (ADEPT) is a two-step approach for the treatment of cancer which seeks to generate a potent cytotoxic agent selectively at a tumor site. In this work described the cytotoxic agent is generated by the action of an enzyme CPG2 on a relatively nontoxic prodrug. The prodrug 1 currently on clinical trial is a benzamide and is cleaved by CPG2 to a benzoic acid mustard drug 1a. We have synthesized a series of new prodrugs 3-8 where the benzamide link has been replaced by, for example, carbamate or ureido. Some of these alternative links have been shown to be good substrates for CPG2 and therefore new candidates for ADEPT. The active drugs 3a and 4a derived from the best of these prodrugs are potent cytotoxic agents (1-2 microM) some 100 times more than 1a. The prodrugs 3 and 4 are some 100-200-fold less cytotoxic, in a proliferating cell assay, than their corresponding active drugs 3a and 4a.

Hypoxia-activated prodrugs as antitumour agents: strategies for maximizing tumour cell killing.

1. Hypoxia arises in solid tumour because of inefficient blood supply. While hypoxic cells are resistant to radiotherapy and probably to many chemotherapeutic drugs they can, in principle, be turned to advantage through the development of hypoxia-activated cytotoxic drugs (bioreductive drugs). 2. Three general approaches to exploiting tumour hypoxia are discussed. The first relies on fluctuating blood flow in tumours and the consequent cycling of cells through the hypoxic compartment. The second incorporates a prodrug approach in which drug activation gives rise to cytotoxic metabolites which diffuse out of hypoxic zones. The third utilizes selective inhibitors of tumour blood flow to induce additional hypoxia and thus enhance bioreductive drug activation. 3. The latter two approaches are illustrated by recent studies with the dinitrobenzamide nitrogen mustard class of bioreductive drugs and their combination with the tumour blood flow inhibitor 5,6-dimethylxanthenone-4-acetic acid.

Bioactivation of dinitrobenzamide mustards by an E. coli B nitroreductase.

A nitroreductase isolated and purified from Escherichia coli B has been demonstrated to have potential applications in ADEPT (antibody-directed enzyme prodrug therapy) by its ability in vitro to reduce dinitrobenzamides (e.g. 5-aziridinyl 2,4-dinitrobenzamide, CB 1954 and its bischloroethylamino analogue, SN 23862) to form cytotoxic derivatives. In contrast to CB 1954, in which either nitro group is reducible to the corresponding hydroxylamine, SN 23862 is reduced by the nitroreductase to form only the 2-hydroxylamine. This hydroxylamine can react with S-acetylthiocholine to form a species capable of producing interstrand crosslinks in naked DNA. In terms of ADEPT, SN 23862 has a potential advantage over CB 1954 in that it is not reduced by mammalian DT diaphorases. Therefore, a series of compounds related to SN 23862 has been synthesized, and evaluated as potential prodrugs both by determination of kinetic parameters and by ratio of IC50 against UV4 cells when incubated in the presence of prodrug, with and without the E. coli enzyme and cofactor (NADH). Results from the two studies were generally in good agreement in that compounds showing no increase in cytotoxicity in presence of enzyme and cofactor were not substrates for the enzyme. None of the analogues were activated by DT diaphorase isolated from Walker 256 carcinoma cells. For those compounds which were substrates for the E. coli nitroreductase, there was a positive correlation between kcat and IC50 ratio. Two compounds showed advantageous properties: SN 25261 (with a dihydroxypropylcarboxamide ring substituent) which has a more than 10-fold greater aqueous solubility than SN 23862 whilst retaining similar kinetic characteristics and cytotoxic potency; and SN 25084, where a change in the position of the carboxamide group relative to the mustard resulted in an increased cytotoxicity ratio and kcat compared with SN 23862 (IC50 ratios 214 and 135; kcat values of 75 and 26.4 sec-1, respectively). An analogue (SN 25507) incorporating both these structural changes had an enhanced kcat of 576 sec-1. This study elucidates some of the structural requirements of the enzyme and aids identification of further directions in the search for suitable prodrugs for an ADEPT nitroreductase system.

Microbial mutagenic effects of the DNA minor groove binder pibenzimol (Hoechst 33258) and a series of mustard analogues.

A series of aniline mustards and half-mustards targeted to DNA by linkage (through a polymethylene chain) to the bisbenzimidazole chromophore of pibenzimol (Hoechst 33258) have been evaluated for their mutagenic properties, as estimated in three strains of Salmonella typhimurium, and for their mitotic crossing-over and petite mutagenesis activities in Saccharomyces cerevisiae strain D5. Agarose gel electrophoresis studies showed that only the derivative with the longest linker chain cross-linked DNA, with the remaining compounds being monoalkylators. The parent (non-alkylator) minor groove binding ligand (Hoechst 33258) was inactive in the bacterial strains TA98 or TA100 but weakly mutagenic in TA102, and caused neither mitotic crossing-over nor 'petite' mutagenesis in yeast. Aniline half-mustard itself (monoalkylator) was an effective base-pair substitution mutagen (events in S. typhimurium strain TA100) with some frameshift mutagenesis activity in TA98, but showed only weak effects in the yeast assays, whereas aniline mustard (cross-linker) was inactive in these bacterial systems but caused substantial amounts of mitotic crossing-over in yeast. The composite molecules studied here showed effects more characteristic of the minor groove binding chromophore than of alkylating moieties. All showed weak mutagenic activity in TA102 and none in TA98. The only compound to show significant mitotic crossing-over ability was the long-chain derivative which cross-linked DNA. For most of the compounds, the mutagenicity data provided no supportive evidence for DNA alkylation. Since other evidence suggests this does occur readily, it is likely to have a different target to that seen with untargeted aniline mustards. The significant antitumor activity and low mutagenic potential shown by these compounds make them worthy of further study.

The use of 32P-postlabelling to detect DNA adducts produced by experimental anticancer drugs: DNA-directed nitrogen mustards.

DNA alkylation by four acridine-linked 'DNA-targeted' aniline mustard derivatives has been studied by 32P-postlabelling. P1 nuclease digestion proved much more efficient than butanol extraction for enhancing the yield of adducted bases for these somewhat hydrophilic compounds. The yield of adducts was maximal after approximately 4 h digestion with micrococcal nuclease/spleen phosphodiesterase and remained relatively constant after that up to 24 h, suggesting that the adducts formed are stable under these conditions. There was some variation in the rates of phosphorylation of the adducts by T4 polynucleotide kinase, with optimal labelling generally occurring after 1 h. The (CH2)5O-linked half-mustard derivative 1 gave five nucleotide 3'-diphosphate adduct spots with calf thymus DNA. Two of these were identified as the adenine N1 and N3 adducts, corresponding to those previously identified as the main base adducts formed by 1 following acid digestion studies. The corresponding full mustard also gave five adduct spots. In contrast, the (CH2)3-linked half-mustard 3 gave only two adduct spots, the most intense of which was identified as a guanine adduct. The corresponding full mustard 4 gave three adduct spots, two of which were identified as guanine adducts. These results agree well with those obtained for the same compounds by the more tedious methods of acid digestion to base adducts, followed by isolation on HPLC, and show that the technique of 32P-labelling can be usefully applied to the study of alkylation of DNA by this class of 'targeted' mustards.

DNA-directed aniline mustards with high selectivity for adenine or guanine bases: mutagenesis in a variety of Salmonella typhimurium strains differing in DNA-repair capability.

Two closely-related aniline monomustards (1 and 2), linked to a DNA-targeting acridine chromophore by a linker chain of different length, show high selectivity for alkylation of polymer DNA. The shorter-chain derivative (2) alkylates mainly at guanine N7 sites, while the longer-chain analogue (1) reacts almost exclusively at adenine N1. The biological effects of these compounds have been studied in standard Ames Salmonella typhimurium strains in order to determine the mutagenic consequences of such well-defined DNA lesions, and the effect of DNA-repair systems on them. Both compounds caused detectable mutations in strains TA1537, TA98 or TA100 and some related strains. Mutation rates were greatly enhanced in strains carrying either a uvrB deletion or the plasmid pKM101. Frameshift mutagenesis by both compounds was completely eliminated by recA deletion, in both the presence or absence of the plasmid. The adenine-selective compound (1) appeared more sensitive to the DNA-repair defects than the guanine-selective derivative (2). Additionally, only the adenine-selective compound (1) caused statistically significant levels of detectable mutation in the repair-proficient strains TA102, TA4001 or TA4006. The bacterial mutagenesis evidence suggests that a bulky, major groove-residing adenine lesion may be more readily recognised by DNA-repair systems, and more likely to lead to a wider range of mutagenic events, than a similar guanine lesion.

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.