Reaction of O6-Benzylguanine-resistant Mutants of Human O6-Alkylguanine-DNA Alkyltransferase with O6-Benzylguanine in Oligodeoxyribonucleotides*

Anthony E. PeggDagger §, Sreenivas KanugulaDagger , Suvarchala EdaraDagger , Gary T. Pauly, Robert C. Moschel, and Karina GoodtzovaDagger

From the Dagger  Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033-0850, and  ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Inactivation of the human DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), by O6-benzylguanine renders tumor cells susceptible to killing by alkylating agents. AGT mutants resistant to O6-benzylguanine can be made by converting Pro140 to an alanine (P140A) or Gly156 to an alanine (G156A). These mutations had a much smaller effect on the reaction with O6-benzylguanine when it was incorporated into a short single-stranded oligodeoxyribonucleotide. Such oligodeoxyribonucleotides could form the basis for the design of improved AGT inhibitors. AGT and mutants P140A and G156A preferentially reacted with O6-benzylguanine when incubated with a mixture of two 16-mer oligodeoxyribonucleotides, one containing O6-benzylguanine and the other, O6-methylguanine. When the 6 amino acids located in positions 159-164 in AGT were replaced by the equivalent sequence from the Escherichia coli Ada-C protein (mutant AGT/6ada) the preference for benzyl repair was eliminated. Further mutation incorporating the P140A change into AGT/6ada giving mutant P140A/6ada led to a protein that resembled Ada-C in preference for the repair of methyl groups, but P140A/6ada did not differ from P140A in reaction with the free base O6-benzylguanine. Changes in the AGT active site pocket can therefore affect the preference for repair of O6-benzyl or -methyl groups when present in an oligodeoxyribonucleotide without altering the reaction with free O6-benzylguanine.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The human DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT),1 is well known to react very rapidly with double-stranded DNA substrates containing O6-methylguanine residues. AGT brings about the transfer of a methyl group from the O6-position of methylated guanines onto a cysteine acceptor located in a PCHR site in the protein sequence (1-4). The S-methylcysteine generated in this reaction is not converted back to cysteine and the protein can therefore act only once. The human AGT is very readily inactivated by exposure to the free base, O6-benzylguanine (5). This inactivation is caused by the ability of O6-benzylguanine to serve as a low molecular weight substrate for the protein forming guanine and an S-benzylcysteine at the active site cysteine-145 residue (6, 7).

O6-Benzylguanine has proven useful as a reagent to examine the importance of AGT activity in protection against the toxic effects of methylating and chloroethylating agents (4). Many studies have shown that treatment of tumor cells in culture and of tumors grown as xenografts in nude mice with O6-benzylguanine to inactivate AGT greatly enhances the killing by both methylating and chloroethylating agents (4, 8-10). Clinical trials testing the effectiveness of O6-benzylguanine to enhance cancer chemotherapy are in progress (9, 11, 12).

The development of resistance to anticancer agents is a major problem in cancer chemotherapy, and the possibility that the generation/selection of mutant forms of AGT resistant to O6-benzylguanine may occur during clinical use is of major importance. Although no such resistance has yet been seen in patients treated with the drug, laboratory studies suggest that AGT variants with reduced activity toward O6-benzylguanine can readily be produced. The first suggestion that this may be the case was provided by the observation that the Escherichia coli protein Ada-C was totally refractory to inhibition by O6-benzylguanine (13). Subsequent studies showed that the yeast AGT and the E. coli Ogt alkyltransferases were also insensitive to O6-benzylguanine (6, 14). Many of the residues of these alkyltransferase proteins are identical. Production of site-directed mutants in the human AGT based on the comparison of these sequences (15-17), and experiments using random mutagenesis (18) showed that several mutations causing single amino acid changes in the AGT were able to greatly reduce the sensitivity to inactivation by O6-benzylguanine. It appears that resistance can be brought about by two types of alteration of the active site pocket: a steric effect reducing the size of the space available at the active site and thus preventing the bulky O6-benzylguanine from binding (16, 19); or the insertion of a hydrophilic residue into the active site, which discourages the binding of the hydrophobic O6-benzylguanine (17).

In the present work, we have compared the ability of the AGT and some mutants to react with O6-benzylguanine when presented either as a free base or when incorporated into a ss oligodeoxyribonucleotide. Several point mutations greatly reducing the ability to react with the free base did not prevent the rapid repair of O6-benzylguanine when it was incorporated into a 16-mer oligodeoxyribonucleotide. These mutations also did not alter the preference of AGT for the repair of benzyl groups rather than methyl groups when incubated with a mixture of 16-mers containing both O6-benzylguanine and O6-methylguanine. Only when multiple alterations conforming to residues present in Ada-C were incorporated was the preference for benzyl repair eliminated.

These results provide further insight into the alkyltransferase reaction and into the design of therapeutically useful inactivators. Relatively short oligodeoxyribonucleotides containing O6-benzylguanine were very effective inhibitors of both the wild type and the mutant AGTs. Therefore, such compounds may form the basis for the design of clinically useful AGT inhibitors that would not be so easily circumvented by single amino acid changes at the active site.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- The synthesis of O6-benzylguanine (5) and the 16-mer oligodeoxyribonucleotides containing O6-benzylguanine or O6-methylguanine were described previously (20). These oligodeoxyribonucleotides were repurified by HPLC (21). The additional oligodeoxyribonucleotides used here were synthesized using methods described previously (21) and were characterized by enzymatic digestion to 2'-deoxyribonucleosides. This analysis agreed within ±10% of the expected values for these oligodeoxyribonucleotides, which were 5'-d(Ab6GC)-3', 5'-d(GAb6GCT)-3', 5'-d(TGAb6GCTG)-3', 5'-d(GTGAb6GCTGT)-3', 5'-d(TGTGAb6GCTGTG)-3' where b6G = O6-benzylguanine. The respective molar extinction coefficients for these oligodeoxyribonucleotides were 2.6, 4.26, 6.16, 7.48, and 9.1 (all × 10-4), respectively.

Labeled O6-benzyl[8-3H]guanine (0.34 mCi/mmol) was prepared by catalytic tritium exchange of O6-benzylguanine with tritiated water by Amersham Pharmacia Biotech and was purified by HPLC (6). N-[3H] Methyl-N-nitrosourea (5.9 mCi/mmol) was obtained from Amersham.

All non-alkylated oligodeoxyribonucleotides were made in the Macromolecular Core Facility, Hershey Medical Center, by using a Milligen 7500 DNA synthesizer. Restriction enzymes were purchased from Life Technologies, Inc. and New England Biolabs (Beverly, MA). Ampicillin, kanamycin, isopropyl beta -D-thiogalactopyranoside, and most other chemicals were purchased from Sigma. Plasmid pGEM-3Zf(+) and T4 polynucleotide kinase were purchased from Promega Corporation (Madison, WI). Pfu DNA polymerase was purchased from Stratagene (La Jolla, CA).

Construction of Plasmid for the Production of Alkyltransferases-- AGT, P140A, G156A, and AGT/6ada were prepared using the pIN vector expression system. The construction of pIN-AGT, pIN-P140A, and pIN-G156A has been described (15, 16, 22). The pIN-AGT/6ada was made by annealing two complementary oligodeoxynucleotides of 80 and 87 base pairs that have the codons for the 6 amino acid sequence from Ada-C (-Arg-Trp-Gly-Val-Ser-Arg-) in place of the codons coding for (-Ser-Gly-Gly-Leu-Ala-Val-) in AGT. These oligodeoxyribonucleotides had the sequences: 5'-GTGGTCTGCAGCAGCGGAGCCGTGGGCAACTACCGCTGGGGCGTGTCGCGTAAGGAATGGCTTCTGGCCCATGAAGGCCA-3' and 3'-TCTCACCAGACGTCGTCGCCTCGGCACCCGTTGATGGCGACCCCGCACAGCGCATTCCTTACCGAAGACCGGGTACTTCCGGTGGCC-5' where the bases that are not identical to the AGT sequence are shown in bold typeface. When annealed, the resulting double-stranded oligodeoxyribonucleotide has protruding ends, which facilitates its ligation into pGEMAGT (16) cut with DraIII and AgeI. The colonies were screened by digestion with NheI since the NheI site is removed for the human AGT sequence by introduction of the altered sequence amino acids from Ada-C protein. The plasmid containing the altered sequence (pGEMAGT/6ada) was digested with BamHI and EcoRI and the 621-base pair fragment ligated into pIN vector cut with these enzymes to form pIN-AGT/6ada as described for pINAGT (22).

The AGT, Ada-C, and P140A/6ada were produced using the pQE30 vector (Qiagen) for the expression of the protein with a small extension (Met-Arg-Gly-Ser-(His)6-Gly-Ser-) at the amino terminus. Construction of the pQE30-AGT (17) and pQE30-Ada-C (23) has been described previously. The pQE30-P140A plasmid was constructed by inserting an EcoNI to DraIII fragment isolated from pGEM-P140A (15) into pQE30-AGT cut with the same enzymes. The pQE30-P140A/6ada plasmid was constructed by inserting the DraIII to AgeI fragment from pINAGT/6ada into pQE30-P140A cut with the same enzymes. All plasmids were verified by sequencing of the entire AGT coding region.

Purification of Alkyltransferase Proteins-- AGT proteins expressed from the pIN vector were purified to homogeneity by ammonium sulfate precipitation, chromatography on Mono-S, and gel filtration as described previously (6, 24). AGT proteins expressed from the pQE30 system were purified using Talon IMAC resin eluted with imidazole as described (17). All proteins used were at least 90% pure as judged by SDS-polyacrylamide gel electrophoresis.

Assay of AGT Sensitivity to Inactivation by O6-Benzylguanine by Oligodeoxyribonucleotides Containing O6-Benzylguanine-- The purified AGT or mutant proteins were incubated with the potential inhibitor in 0.1 ml of 50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 5.0 mM dithiothreitol, 10 µg of hemocyanin or 10 µg of calf thymus DNA for 30 min at 37 °C. The residual alkyltransferase activity was then determined by a 30-min incubation period with a 3H-methylated DNA substrate, which had been methylated by reaction with N-[3H]methyl-N-nitrosourea essentially as described (5, 25). The volume was increased to 1.0 ml during the second incubation. The results were expressed as the percentage of the AGT activity remaining and then used to calculate the ED50 value for the inhibitor where the ED50 is the concentration needed to reduce the AGT activity by 50% in a 30-min incubation period at 37 °C. There was no significant loss (<5%) of AGT activity when no inhibitor was added. The concentrations of the oligodeoxyribonucleotides containing O6-benzylguanine were determined using the extinction coefficients at 260 nm.

Rate of Formation of Guanine from O6-Benzylguanine by AGT-- Measurements of [8-3H]guanine formation from O6-benzyl[8-3H]guanine were carried out using an assay mixture consisting of 50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, and 5 mM dithiothreitol in a volume of 0.25 ml. After incubation for periods of up to 60 min, the formation of [8-3H]guanine was determined by HPLC (26). Results were expressed as cpm of [8-3H]guanine formed per µg of protein per min.

Assay of Dealkylation of Oligodeoxyribonucleotides Containing O6-Methylguanine or O6-Benzylguanine-- The purified alkyltransferase proteins were incubated in 50 mM Tris-HCl, pH 7.5, 0. 5 mM dithiothreitol, and 0.1 mM EDTA with 5'-d(AACAGCCATATa6GGCCC)-3' where a6G represents O6-benzylguanine or O6-methylguanine. The reaction was stopped by the addition of 1% SDS, and the mixture of oligodeoxyribonucleotides was separated by HPLC. The separation was carried out on a Beckman C18 5-µm Ultrasphere ODS reverse-phase column (4.6 × 250 mm) with a Brownlee RP-300 Aquapore 7-µm precolumn (0.46 × 300 mm) at 45 °C using a flow rate of 2 ml/min and a linear gradient of increasing methanol at 0.23% per min over 30 min starting from 14% methanol in 50 mM sodium phosphate, pH 6.3. The retention times of the unalkylated, methylated and benzylated oligodeoxyribonucleotides that were detected by absorbance at 254 nm were 9.5, 15, and 21 min, respectively.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Mutations in AGT Affecting Reaction with O6-Benzylguanine-- Previous studies have identified a number of single amino acid mutations in the human AGT sequence that impart resistance to O6-benzylguanine. These include the alterations of Pro138 or Pro140 to Ala or Lys (15, 16) and the alteration of Gly156 to Ala or Trp (16). Conversely, the E. coli Ada-C protein is totally resistant to O6-benzylguanine but can be rendered sensitive by placing a Pro residue in place of Ala at position 316 (which is equivalent to 140 in AGT) and by converting a bulky Trp336 residue, which blocks access to the active site, to Ala (23, 27). These residues are underlined in Fig. 1, which also shows the sequence of the AGT mutant proteins used in the experiments described here.


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Fig. 1.   Sequence containing the active site of AGT, P140A, G156A, AGT/6ada, P140A/6ada, and Ada-C. Residues conserved in all of these alkyltransferase sequences are shown in bold typeface. The mutations in the AGT sequence are shown underlined, and the residues referred to in the Ada-C sequence are shown in underlined italic typeface.

These findings support the concept that a Pro residue at position 140 is needed for O6-benzylguanine to react well with the AGT. However, the presence of a Trp residue in the active site region of the AGT at the position presumably equivalent to Trp336 was not sufficient to prevent good reaction with O6-benzylguanine since the mutant AGT/6ada was not markedly resistant to inactivation by O6-benzylguanine (Table I) and produced almost the same amount of [8-3H]guanine when allowed to react with O6-benzyl[8-3H]guanine as the wild type AGT (Table I). The AGT/6ada protein has 6 amino acids located in positions 159-164 replaced by the equivalent sequence from Ada-C, which includes Trp336 (Fig. 1). Moreover, when the P140A mutation was combined with AGT/6ada mutation to form P140A/6ada, the resistance to O6-benzylguanine inactivation and the rate of [8-3H]guanine production was not significantly different from that of the P140A alone (Table I).

                              
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Table I
Inactivation of alkyltransferase activity by O6-benzylguanine and production of guanine from O6-benzylguanine by alkyltransferases

As indicated in Table I, the P140A, G156A, and P140A/6ada AGT mutant proteins were much less able to react with O6-benzylguanine as a free base as demonstrated by the rate of [8-3H]guanine formation from O6-benzyl[8-3H]guanine. However, it is known that the rate of alkyl group transfer by AGT or Ada-C is enhanced by binding DNA, which causes a conformational change in the protein exposing and activating the cysteine at the alkyl acceptor site (26, 27). In our studies, the presence of calf thymus DNA increased the rate of [8-3H]guanine formation with all of the mutants and had a larger effect on those containing a mutation at Pro140, but even in the presence of DNA, the P140A and G156A mutations greatly reduced the rate of the AGT reaction with O6-benzylguanine (Table I).

Ability of AGT and Mutants to React with O6-Benzylguanine in Oligodeoxyribonucleotides-- All of these mutant proteins were able to react very rapidly with O6-benzylguanine when it was incorporated into a 16-mer oligodeoxyribonucleotide with reaction being complete with less than 5 min (results not shown).

In agreement with previous reports (23), when the wild type AGT was incubated with an equimolar mixture of 16-mers of identical sequence containing either O6-benzylguanine or O6-methylguanine, the AGT slightly preferred the benzylated substrate (Fig. 2). This preference was also the case with both the G156A and the P140A mutant AGT proteins (Fig. 2). However, the substitution of the positions 159-164 with the 6 amino acids from Ada (mutant AGT/6ada) led to a slight preference for the methylated 16-mer, and when this alteration was combined with the P140A substitution (mutant P140A/6ada), this led to a marked preference for repair of the methylated substrate (Fig. 2). The P140A/6ada mutant AGT therefore resembled Ada-C, which as previously reported (23) strongly prefers the methylated substrate.


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Fig. 2.   Competition for repair of O6-methylguanine and O6-benzylguanine in 16-mer oligodeoxyribonucleotides by AGT, G156A, P140A, AGT/6ada, P140A/6ada, and Ada-C. The first column shows results for AGT (0, 20, 40, and 60 µg), the second column shows results for G156A (0, 20, 40, and 60 µg), the third column shows results for P140A (0, 30, 50, and 60 µg), the fourth column shows results for AGT/6ada, (0, 20, 40, and 80 µg), the fifth column shows results for P140A/6ada (0, 20, 40 and 60 µg), and the sixth column shows results for Ada-C (0, 25, 50, and 200 µg). The proteins were incubated with a mixture of 600 pmol each of 5'-d(AACAGCCATATm6GGCCC)-3' and 5'-d(AACAGCCATATb6GGCCC)-3' in a total volume of 0.05 ml for 10 min at 37 °C. The products were then separated by HPLC as described under "Experimental Procedures." The arrow marked A coincides with the elution of an authentic marker of 5'-d(AACAGCCATATGGCCC)-3', the arrow marked B coincides with the elution of 5'-d(AACAGCCATATm6GGCCC)-3', and the arrow marked C coincides with the elution of 5'-d(AACAGCCATATb6GGCCC)-3'.

Inactivation of O6-Benzylguanine-resistant Mutants by Reaction with Oligodeoxyribonucleotides Containing O6-Benzylguanine-- Incubation of control, P140A, or G156A AGT with oligodeoxyribonucleotides containing O6-benzylguanine led to a loss of AGT activity (Fig. 3 and Table II). All of the oligodeoxyribonucleotides tested were more potent than O6-benzylguanine itself or O6-benzyl-2'-deoxyguanosine (which we have previously shown to actually be less active than the free base (28)). Even a 3-mer was more active than the free base but maximal effects were seen with oligodeoxyribonucleotides containing 5-9 nucleotides. It should be noted that although the reduction in the ED50 value for the mutants was much greater than for the wild type (by a factor of >1000 for G156A and by a factor of 60-100 for P140A compared with a factor of 15-30 for control AGT), there was still a 4-11-fold difference in the ED50 values between the mutant AGT proteins and the control AGT.


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Fig. 3.   Inactivation of AGT, G156A, and P140 by incubation with oligodeoxyribonucleotides containing O6-benzylguanine. The AGT (upper panel), G156A mutant (middle panel), or P140A mutant proteins were incubated at 37 °C in 0.1 ml of solution containing 50 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 0. 1 mM EDTA, and the concentration shown of oligodeoxyribonucleotides containing 3, 5, 7, 9, or 11 nucleotides with a central O6-benzylguanine residue (see "Experimental Procedures" for sequences), and the remaining alkyltransferase activity measured as described under "Experimental Procedures."

                              
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Table II
Inactivation of AGTs by reaction with O6-benzylguanine, O6-benzyl-2'-deoxyguanosine or oligodeoxyribonucleotides containing O6-benzylguanine

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

We have demonstrated previously that when presented with a mixture of ss oligodeoxyribonucleotide substrates, the human AGT repairs O6-benzylguanine preferentially over O6-methylguanine, whereas the E. coli Ada-C alkyltransferase exhibits a strong preference to act on O6-methylguanine (23). The results presented here show that this preference is primarily due to the amino acids at positions between the conserved residues, Tyr-158 and Lys-165. Replacing the human sequence at this region with the sequence from Ada-C abolished the preference for benzyl repair (Fig. 2). A secondary contributor to the preference is the presence of Pro at position 140 in the human AGT. Mutation of this residue to Ala to conform to the Ada-C sequence did not by itself alter the specificity of alkyl repair using the 16-mer ss oligodeoxyribonucleotide substrates. However, when this alteration was combined with the insertion of the 6 amino acids from Ada-C between positions 158 and 165, the AGT protein showed a strong preference for methyl repair (Fig. 2). These results can be explained by the alteration in the active site pocket for the AGT. Several authors have suggested that AGT resembles a number of other DNA repair proteins in flipping the nucleotide to be repaired out of the DNA helix (29-32). This mechanism requires the "flipped" base to bind into an active site pocket. This pocket in AGT must contain the Cys-145 acceptor site and be able to accommodate the bulky and hydrophobic benzyl group. The replacement of the residues SGGLAV with RWGVSR is likely to both reduce the space available and to increase the hydrophilicity of the protein region forming this pocket. These alterations would reduce the ability to repair O6-benzyl groups. The conversion of the preceding Pro140 residue to Ala changes the orientation of the peptide chain containing this 6-amino acid sequence and could position it in a way to further favor methyl repair.

A second major conclusion from our results is that the structural features responsible for the good interaction of the wild type human AGT with O6-benzylguanine as a free base are not identical to those for repair of O6-benzylguanine in ss oligodeoxyribonucleotides. Observations supporting this argument include: (a) the fact that the P140A and G156A mutant AGTs, which have greatly reduced rate of inactivation by free O6-benzylguanine (16), resembled the wild type AGT in showing a preference for benzyl rather than methyl repair of 16-mer oligodeoxyribonucleotides substrates (Fig. 2); (b) the replacement of the SGGLAV with RWGVSR to form AGT/6ada or P140A/6ada did not reduce reaction with O6-benzylguanine as a free base (Table I) but did confer a preference for repair of methyl rather than benzyl in 16-mer oligodeoxyribonucleotide substrates (Fig. 2); and (c) the finding that inactivation of the P140A and G156A mutant AGTs by O6-benzylguanine-containing oligodeoxyribonucleotide substrates containing from 5 to 11 nucleotides required, respectively, only 4-11-fold higher concentrations of the inhibitor than the wild type as opposed to 25- and 300-fold higher concentrations of free O6-benzylguanine (Fig. 3 and Table II). These results are in agreement with a recent report that AGT can readily be inactivated by reaction with 25-mer substrates containing a central O6-benzyl adduct including O6-(2-fluorobenzyl)guanine and O6-benzylhypoxanthine, which are very poor inactivators of AGT when used as the free base (33). They are also consistent with our previous study showing that E. coli Ada-C can react, albeit slowly, with a 16-mer containing O6-benzylguanine (23) although no reaction with O6-benzylguanine as a free base could be detected (6).

Several factors may contribute to the discrepancy in the AGT reactions with O6-benzylguanine when presented as a free base and as a component of an oligodeoxyribonucleotide. A major influence is likely to be that the binding of the oligodeoxyribonucleotide is mediated predominantly by the DNA binding domain of the AGT. Although this domain has not been fully defined, studies based on the crystal structure of Ada-C and studies of mutations affecting DNA binding suggest that it is located in the helix-turn-helix region, which resides between amino acids Phe-94 and Asn-137 (24, 27, 34). This region is not altered in the O6-benzylguanine-resistant mutants, and therefore the binding of oligodeoxyribonucleotides containing O6-benzylguanine is unlikely to be affected greatly. Furthermore, binding is likely to present the O6-benzyl adduct in the DNA in a manner favoring transfer to the cysteine acceptor site. The limiting factor in the reaction of the free base, O6-benzylguanine, is likely to be the initial binding, which is very weak since the Kd for binding is more than 1 mM.2 Mutations reducing the ability of O6-benzylguanine to bind either by restricting the size of the binding pocket or by altering the largely hydrophobic face of this pocket may therefore cause a much greater decrease in the rate of benzyl transfer from the free base than from an oligodeoxyribonucleotide. A secondary factor that may also contribute to the better reaction with O6-benzylguanine in ss oligodeoxyribonucleotides may be that the alkyltransferase protein undergoes a conformational change on binding DNA (24, 26, 27, 34, 35). This conformational change may expose and activate the cysteine acceptor group and may also alter the size of the active site pocket and thus allow better reaction with a bulky substrate.

The development of drug resistance is a major problem in cancer chemotherapy. The ability of a number of different point mutations in the AGT sequence to give rise to resistance to O6-benzylguanine (16-18) therefore raises considerable concern that such resistant forms may arise, or be selected for, during the course of chemotherapy with methylating or chloroethylating agents. This may necessitate the development of additional AGT inhibitors able to inactivate these resistant forms. Phase I clinical trials of O6-benzylguanine indicate that plasma levels of the drug or active metabolites are unlikely to exceed 25 µM (9) and this will not be adequate to inactivate known resistant AGT mutants. Short oligodeoxyribonucleotides containing O6-benzylguanine may serve as the basis for the design of such agents. The results shown in Table II indicate that the rate of AGT reaction with oligodeoxyribonucleotides containing a central O6-benzylguanine increases with oligodeoxyribonucleotide length but that a maximal rate only requires a 5-9-mer. This finding is in agreement with previous studies on the use of ss oligodeoxyribonucleotides containing O6-methyl or ethyl adducts as AGT substrates (36, 37) and with studies of oligodeoxyribonucleotide binding using biophysical techniques that suggest that the AGT contacts about 8 base pairs (35, 38, 39). Since the ED50 values for inactivation of AGT by such 5-9-mers were of the order of 7-60 nM (Table II), it should be possible to obtain adequate inhibition of the resistant mutants even though the O6-benzylguanine-resistant AGT mutants were nearly an order of magnitude less sensitive to the oligodeoxyribonucleotides than the wild type. This approach should therefore prove useful for the design of therapeutically valuable AGT inhibitors.

    FOOTNOTES

* This research was supported by Grants CA-18138, CA-57725, and CA-71976 from the National Cancer Institute (to A. E. P.) and by National Cancer Institute Contract with ABL (to R. C. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, P. O. Box 850, 500 University Dr., Hershey, PA 17033-0850. Tel.: 717-531-8152; Fax: 717-531-5157; E-mail: aepl{at}psu.edu.

1 The abbreviations used are: AGT, human O6-alkylguanine-DNA alkyltransferase; Ada-C, the carboxyl-terminal domain of the E. coli O6-alkylguanine-DNA alkyltransferase; ss, single-stranded; HPLC, high performance liquid chromatography.

2 A. E. Pegg, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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