Biomedical subjects

A E Pegg

Publications and source records attributed to A E Pegg.

At least 19 recordsLinked to original sources

PubMed1999

Reaction and binding of oligodeoxynucleotides containing analogues of O6-methylguanine with wild-type and mutant human O6-alkylguanine-DNA alkyltransferase.

O6-Alkylguanine-DNA alkyltransferase (AGT) repairs DNA by transferring the methyl group from the 6-position of guanine to a cysteine residue on the protein. We previously found that the Escherichia coli Ada protein makes critical interactions with O6-methylguanine (O6mG) at the N1- and O6-positions. Human AGT has a different specificity than the bacterial protein. We reacted hAGT with double-stranded pentadecadeoxynucleotides containing analogues of O6mG. The second-order rate constants were in the following order (x10(-)5 M-1 s-1): O6mG (1.4), O6-methylhypoxanthine (1.6) > Se6-methyl-6-selenoguanine (0.1) > S6-methyl-6-thioguanine (S6mG) (0.02) >> S6-methyl-6-thiohypoxanthine (S6mH), O6-methyl-1-deazaguanine (O6m1DG), O6-methyl-3-deazaguanine (O6m3DG), and O6-methyl-7-deazaguanine (O6m7DG) (all <0.0001). Electrophoretic mobility shift assays were carried out to determine the binding affinity to hAGT. Oligodeoxynucleotides containing O6mG, S6mG and O6m3DG bound to AGT in the presence of competitor DNA with Kd values from 5 to 20 microM, while those containing G, S6mH, O6m1DG, and O6m7DG did not (Kd > 200 microM). These results indicate that the 1-, N2-, and 7- positions of O6mG are critical in binding to hAGT, while the 3- and O6-positions are involved in methyl transfer. These results suggest that the active site of ada AGT is more flexible than hAGT and may be the reason ada AGT reacts with O4mT faster than hAGT.

Binding Sites↗

PubMed1999

Alteration of the conserved residue tyrosine-158 to histidine renders human O6-alkylguanine-DNA alkyltransferase insensitive to the inhibitor O6-benzylguanine.

The DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) protects cells from alkylation damage. O6-Benzylguanine (BG) is a potent inactivator of human AGT (ED50 of 0.1 microM) that is currently undergoing clinical trials to enhance chemotherapy by alkylating agents. In a screen of AGT mutants randomly mutated at position glycine-160, we found that the double mutant Y158H/G160A protected Escherichia coli from killing by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) even in the presence of BG and that the AGT activity of this mutant was strongly resistant to BG (ED50 of 180 microM). Because the single mutant G160A was not resistant to BG, this suggested that the presence of the charged histidine residue at position 158 was responsible. This hypothesis was confirmed by the construction of the single mutation Y158H. The Y158H-mutant AGT was slightly less active than wild-type AGT for the repair of methylated DNA in vitro, but it protected E. coli from killing by MNNG even in the presence of BG and had an ED50 for the inactivation by BG of 620 microM. In contrast, mutant Y158F had an ED5o of 0.2 microM. Previous studies (M. Xu-Welliver et al., Cancer Res., 58: 1936-1945, 1998) have shown that mutant P140K is highly resistant to BG (ED50 of >1200 microM). Models of human AGT suggest that the side chain of the lysine inserted into this mutant is close to tyrosine-158 and that the positively charged lysine side-chain may interfere with BG binding. The double mutants P140K/Y158H and P140K/Y158F resembled P140K and Y158H in being highly resistant to BG, but the use of a sensitive assay for reaction of BG with AGT indicated that their abilities to react were in the order P140K/ Y158H < P140K < P140K/Y158F. These results confirm that the presence of a positively charged residue close to the active site of human AGT renders it highly resistant to BG without substantially affecting activity toward methylated DNA substrates. Such mutants may limit the value of BG therapy if they arise in malignant cells during chemotherapy, but the mutant sequences may be useful for gene therapy approaches in which BG-resistant human AGTs are used to prevent hematopoietic toxicity. At least 28 AGT sequences (from 25 species) have now been described. In 25 of these, the position equivalent to 158 in the human AGT is also a tyrosine, and in the other 3, it is a phenylalanine. The importance of an aromatic ring side chain at this position is emphasized by previous studies (S. Edara et al., Carcinogenesis, 16: 1637-1642, 1995), which show that the replacement by alanine renders human AGT inactive. Our results show that histidine can also substitute for tyrosine at this position.

Enzyme Inhibitors↗

PubMed1999

Properties and regulation of human spermidine/spermine N1-acetyltransferase stably expressed in Chinese hamster ovary cells.

Spermidine/spermine N1-acetyltransferase (SSAT) appears to be the rate-limiting enzyme of polyamine catabolism, yet studies of its regulation have been limited by the low amounts of SSAT in uninduced cells. A system for studying SSAT was established by stably transfecting Chinese hamster ovary cells with a construct where SSAT cDNA was under control of the cytomegalovirus promoter. Thirteen of 44 clones expressed significantly increased SSAT activity (650-1900 compared with 24 pmol/min/mg protein in control cells). SSAT activity was directly proportional to SSAT protein, which turned over very rapidly (t(1)/(2) of 29 min) and was degraded through the ubiquitin/proteasomal pathway. The increased SSAT activity caused perturbations in polyamine homeostasis and led to a reduction in the rate of growth under clonal conditions. N1,N12-bis(ethyl)spermine greatly increased SSAT activity in controls and SSAT transfected clones (to about 10 and 60 nmol/min/mg protein, respectively). N1, N12-Bis(ethyl)spermine caused an increase in the SSAT half-life and a slight increase in SSAT mRNA, but these changes were insufficient to account for the increase in SSAT protein suggesting that translational regulation of SSAT must also occur.

Acetyltransferases↗

PubMed1999

Role of cysteine-82 in the catalytic mechanism of human S-adenosylmethionine decarboxylase.

S-Adenosylmethionine decarboxylase is a pyruvate-dependent enzyme. The enzyme forms a Schiff base with substrate, S-adenosylmethionine, through the pyruvoyl moiety. This facilitates the release of CO2 from the substrate, which must then be protonated on the alpha carbon in order to permit hydrolysis of the Schiff base to release the product. The catalytic mechanism of human S-adenosylmethionine decarboxylase was investigated via mutagenic and kinetic approaches. The results of enzyme kinetic studies indicated that Cys-82 is a crucial residue for activity and this residue has a basic pKa. Iodoacetic acid inhibited wild-type enzyme activity in a time- and pH-dependent manner but did not affect the already reduced activity of mutant C82A. Reaction of this mutant with iodoacetic acid led to approximately one less mole of reagent being incorporated per mole of enzyme alphabeta dimer than with wild-type S-adenosylmethionine decarboxylase. Both wild-type and C82A mutant S-adenosylmethionine decarboxylases were inactivated by substrate-mediated transamination, but this reaction occurred much more frequently with C82A than with wild-type enzyme. A major proportion of the recombinant C82A mutant protein was in the transaminated form in which the pyruvoyl cofactor is converted into alanine. This suggests that incorrect protonation of the pyruvate, rather than the substrate, occurs much more readily when Cys-82 is altered. On the basis of these results, it was postulated that residue Cys-82 may be the proton donor of the decarboxylation reaction catalyzed by S-adenosylmethionine decarboxylase.

Adenosylmethionine Decarboxylase↗

PubMed1999

Modulation of cyclophosphamide activity by O6-alkylguanine-DNA alkyltransferase.

PURPOSE: The human medulloblastoma cell line D283 Med (4-HCR), a line resistant to 4-hydroperoxycyclophosphamide (4-HC), displays enhanced repair of DNA interstrand crosslinks induced by phosphoramide mustard. D283 Med (4-HCR) cells are cross-resistant to 1,3-bis(2-chloroethyl)- -nitrosourea, but partial sensitivity is restored after elevated levels of O6-alkylguanine-DNA alkyltransferase (AGT) are depleted by O6-benzylguanine (O6-BG). Studies were conducted to define the activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide against D283 Med (4-HCR) after AGT is depleted by O6-BG. METHODS: Limiting dilution and xenograft studies were conducted to define the activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide with or without O6-BG. RESULTS: The activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide against D283 Med (4-HCR) was increased after AGT depletion by O6-BG preincubation. Similar studies with Chinese hamster ovary cells, with or without stable transfection with a plasmid expressing the human AGT protein, revealed that the AGT-expressing cells were significantly less sensitive to 4-HC and 4-hydroperoxydidechlorocyclophosphamide. Reaction of DNA with 4-HC, phosphoramide mustard, or acrolein revealed that only 4-HC and acrolein caused a decrease in AGT levels. CONCLUSIONS: We propose that a small but potentially significant part of the cellular toxicity of cyclophosphamide in these cells is due to acrolein, and that this toxicity is abrogated by removal of the acrolein adduct from DNA by AGT.

Animals↗

PubMed1999

Translational regulation of ornithine decarboxylase and other enzymes of the polyamine pathway.

It has long been known that polyamines play an essential role in the proliferation of mammalian cells, and the polyamine biosynthetic pathway may provide an important target for the development of agents that inhibit carcinogenesis and tumor growth. The rate-limiting enzymes of the polyamine pathway, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), are highly regulated in the cell, and much of this regulation occurs at the level of translation. Although the 5' leader sequences of ODC and AdoMetDC are both highly structured and contain small internal open reading frames (ORFs), the regulation of their translation appears to be quite different. The translational regulation of ODC is more dependent on secondary structure, and therefore responds to the intracellular availability of active eIF-4E, the cap-binding subunit of the eIF-4F complex, which mediates translation initiations. Cell-specific translation of AdoMetDC appears to be regulated exclusively through the internal ORF, which causes ribosome stalling that is independent of eIF-4E levels and decreases the efficiency with which the downstream ORF encoding AdoMetDC protein is translated. The translation of both ODC and AdoMetDC is negatively regulated by intracellular changes in the polyamines spermidine and spermine. Thus, when polyamine levels are low, the synthesis of both ODC and AdoMetDC is increased, and an increase in polyamine content causes a corresponding decrease in protein synthesis. However, an increase in active eIF-4E may allow for the synthesis of ODC even in the presence of polyamine levels that repress ODC translation in cells with lower levels of the initiation factor. In contrast, the amino acid sequence that is encoded by the upstream ORF is critical for polyamine regulation of AdoMetDC synthesis and polyamines may affect synthesis by interaction with the putative peptide, MAGDIS.

Adenosylmethionine Decarboxylase↗

PubMed1999

Synthesis of DNA oligonucleotides containing site-specifically incorporated O6-[4-oxo-4-(3-pyridyl)butyl]guanine and their reaction with O6-alkylguanine-DNA alkyltransferase.

DNA pyridyloxobutylation occurs during the metabolic activation of the tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). This pathway contributes significantly to the carcinogenic and mutagenic activity of these nitrosamines. In general, the chemical structure of pyridyloxobutyl adducts are not well understood. Recently, an AGT reactive pyridyloxobutyl adduct was identified as O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobG). To better understand the importance of this adduct to the biological activity of pyridyloxobutylating agents, we developed a method for site-specifically incorporating O6-pobG into DNA oligonucleotides. They were synthesized using the phosphoramidite of the precursor 2'-deoxy-O6-{3-[2-(3-pyridyl)-1,3-dithian-2-yl]propyl}guanosine. The dithiane group was oxidatively removed with N-chlorosuccinimide in a final postoligomerization reaction to generate the desired product. Human AGT with a polyhistidine tag was able to repair the O6-pobG-containing DNA oligonucleotide, generating unmodified oligonucleotide. These results are consistent with an alkyl group transfer mechanism for the repair of O6-pobG by AGT.

DNA↗

PubMed1999

Expression of the inactive C145A mutant human O6-alkylguanine-DNA alkyltransferase in E.coli increases cell killing and mutations by N-methyl-N'-nitro-N-nitrosoguanidine.

Human O6-alkylguanine-DNA alkyltransferase (AGT) counteracts the mutagenic and toxic effects of methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by removing the methyl group from O6-methylguanine lesions in DNA. The methyl group is transferred to a cysteine acceptor residue in the AGT protein, which is located at residue 145. The C145A mutant of AGT in which this cysteine is converted to an alanine residue is therefore inactive. When this C145A mutant was expressed in an Escherichia coli strain lacking endogenous alkyltransferase activity, the number of G:C-->A:T mutations actually increased and the toxicity of the MNNG treatment was enhanced. These effects were not seen when an E.coli strain also lacking nucleotide excision repair (NER) was used. The enhancement of mutagenesis and toxicity of MNNG produced by the C145A mutant AGT was not seen with another inactive mutant Y114E that contains a mutation preventing DNA binding, and the double mutant C145A/Y114E was also ineffective. These results suggest that the C145A mutant AGT binds to O6-methylguanine lesions in DNA and prevents their repair by NER. The inactive C145A mutant AGT also increased the number of A:T-->G:C transition mutations in MNNG-treated cells. These mutations are likely to arise from the minor methylation product, O4-methylthymine. However, expression of wild-type AGT also increased the incidence of these mutations. These results support the hypothesis that mammalian AGTs bind to O4-methylthymine but repair the lesion so slowly that they effectively shield it from more efficient repair by NER.

Amino Acid Substitution↗

PubMed1999

Leukocyte O6-alkylguanine-DNA alkyltransferase from human donors is uniformly sensitive to O6-benzylguanine.

O6-Alkylguanine-DNA alkyltransferase (AGT) is the key DNA repair protein responsible for resistance to chloroethylating and methylating agents that attack at the O6 position of guanine. O6-Benzylguanine (BG), a potent inhibitor of AGT, has recently entered clinical trials. A number of point mutations and at least one human polymorphism within AGT are associated with AGT resistance to inactivation by BG. In this study, we evaluated AGT inhibition by BG in an in vitro assay of peripheral blood mononuclear cell AGT from 56 normal donors, 42 Caucasians, and 14 Japanese. AGT activity ranged from 2.7 to 21.9 fmol/microg DNA and was similar in Japanese and Caucasian donors. Depletion of AGT by BG was uniform in all donors with mean ED50s of 037 microM BG in Caucasians and 0.36 microM BG in Japanese. To determine whether the gly160arg AGT polymorphism described in the Japanese population, and recently shown to be BG resistant, could be detected by this assay, we mixed purified gly160arg AGT protein with blood mononuclear cell extract and measured in vitro BG inactivation. The ED50 for the mixture of the gly160arg AGT and mononuclear cell extract was 9 microM BG. On the basis of results in 56 donors, we conclude that BG-resistant AGT, defined as an ED50 in mononuclear cells of >1 microM BG, is present in 0 of 56 donors, (95% confidence interval, 0-6%), suggesting that polymorphisms producing AGT-resistant BG are unusual in humans.

Alkyl and Aryl Transferases↗

PubMed1998

Alpha-difluoromethylornithine inhibits N-nitrosomethylbenzylamine-induced esophageal carcinogenesis in zinc-deficient rats: effects on esophageal cell proliferation and apoptosis.

Sustained, increased cell proliferation induced by dietary zinc deficiency in rats plays a critical role in esophageal carcinogenesis. It is the determining factor that converts an otherwise nontumorigenic dose of N-nitrosomethylbenzylamine (NMBA) into a highly tumorigenic one. We studied whether the increased esophageal cell proliferation and susceptibility to NMBA-induced carcinogenesis induced by zinc deficiency can be inhibited by alpha-difluoromethylornithine (DFMO), an enzyme-activated, irreversible inhibitor of ornithine decarboxylase (the first enzyme in polyamine synthesis). Weanling rats were divided into four groups: Zn+/DFMO-, Zn+/DFMO+, Zn-/DFMO-, and Zn-/DFMO+. They were fed ad libitum either a zinc-sufficient (Zn+, 75 ppm zinc) or a zinc-deficient (Zn-, 4 ppm zinc) diet and given either deionized water (DFMO-) or 1% DFMO in deionized water (DFMO+). After 5 weeks, 5-19 animals from each group were sacrificed after in vivo 5-bromo-2'-deoxyuridine labeling to detect cells in S phase. The remaining animals in each group were given a single intragastric dose of NMBA at 2 mg/kg and sacrificed 12 weeks later for tumor incidence analysis. At week 5, DFMO treatment greatly decreased (by 48-82%) the levels of putrescine and spermidine in rat esophagus, colon, and liver, irrespective of dietary zinc intake. The increased esophageal cell proliferation induced by dietary zinc deficiency, as measured by the labeling index, the number of labeled cells, and the total number of cells, was substantially reduced by DFMO. This was accompanied by an increase in the rate of apoptosis. In addition, the expression of bax protein, an apoptosis accelerator, was markedly stronger in esophagi from Zn-/DFMO+ animals that showed increased apoptosis, whereas increased expression of bcl-2, an inhibitor of apoptosis, was only seen in the highly proliferative, zinc-deficient esophagus (Zn-/DFMO-). At week 12 after NMBA dosing, DFMO reduced the incidence of esophageal tumors from 80 to 4% in zinc-deficient rats. Our data showed that DFMO effectively inhibited the increased esophageal cell proliferation induced by dietary zinc deficiency and reduced the incidence of esophageal tumors induced by a single dose of NMBA in zinc-deficient animals. Our results also indicate a role for increased apoptosis in the mechanism(s) whereby DFMO brings about the inhibition of cell proliferation and tumor induction. These findings support a role for DFMO as a chemopreventive agent.

Animals↗

PubMed1998

Investigation of the role of tyrosine-114 in the activity of human O6-alkylguanine-DNA alkyltranferase.

Tyrosine-114 is one of 13 totally conserved amino acids in all known sequences of O6-alkylguanine-DNA alkyltransferase (AGT). The importance of this amino acid in repair of alkylated DNA by AGT was studied by changing it to phenylalanine (F), alanine (A), threonine (T), or glutamic acid (E) in human AGT. The activities of the mutant proteins were then compared to those of the wild type with regard to abilities to do the following: (a) protect Escherichia coli from the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); (b) repair methylated DNA in vitro; (c) bind to oligodeoxynucleotides containing O6-methylguanine; and (d) react with the low molecular weight pseudosubstrate, O6-benzylguanine. When expressed at high levels in E. coli strain GWR109, lacking endogenous AGT, the wild type and the Y114F mutant were highly effective in reducing mutations and cell killing by MNNG. The Y114A mutant had a much smaller protective effect, and mutants Y114T and Y114E were inactive. Purified preparations of all four AGT mutants showed an approximately similar degree (74-120-fold) of reduction in the rate of reaction with O6-benzylguanine. In contrast, the degree of reduction in activity toward methylated DNA substrates in vitro varied according to the mutation with the more conservative Y114F producing only a 30-fold reduction and the most drastic change of Y114E abolishing activity completely. Alteration Y114A produced a 1000-fold reduction whereas Y114T reduced activity by 10000-fold. All of the mutations affected the binding of AGT to single- or double-stranded oligodeoxynucleotides containing O6-methylguanine. The extent of increase in the Kd varied according to the amino acid with 2-5-fold (F), 7-11-fold (A), 167-200-fold (T), and 600-1000-fold (E) increases. These results are consistent with tyrosine-114 playing a role both in the binding of AGT to its DNA substrate and in facilitating the transfer of the alkyl group. It is probable that AGT resembles other DNA repair proteins in bringing about a "flipping out" of the target base from the DNA helix. Tyrosine-114 is therefore an excellent candidate for a key role in the interaction with the flipped O6-methylguanine. The results also show that when large amounts of AGT are produced in the cell, substantial decreases in the efficiency with which AGT can repair methylated DNA do not prevent the ability to protect E. coli from toxic alkylating agents. Mutant Y114F, whose activity was reduced by 30-fold, was equal to wild-type AGT in bringing about this protection.

Alanine↗

PubMed1998

Structure/function relationship studies on the T/S residues 173-177 of rat ODC.

A well-conserved T/S cluster was detected among vertebrate ornithine decarboxylase by computer analysis (E. Viguera, O. Trelles, J.L. Urdiales, J.M. Matés, F. Sánchez-Jiménez, Trends Biochem. Sci. 19 (1994) 318-319). In the present report we studied the role of these residues (173, 176 and 177 in rat ornithine decarboxylase (ODC)) in enzymic activity and stability by in vitro expression, kinetic characterization and in vitro degradation of site-directed mutants. These T/S residues are substituted by a D/E-enriched fragment in other lower eukaryotic ODCs. The substitution of the T/S-enriched fragment (TLKTS) of rat ODC by the negative charged fragment of T. brucei ODC (KVEDC) did not affect protein stability, but increased Km values of the mutant enzyme. The substitution of the T/S residues by alanine also has a similar effect on rat ODC kinetic values. However, results indicate that polarity of the fragment must be an important factor for protein conformation, since the latter mutant, having no T/S or D/E residue in the fragment (ALKAA), showed reduced stability in vitro.

Amino Acid Sequence↗

PubMed1998

Ornithine decarboxylase induction in transformation by H-Ras and RhoA.

The objective of these studies has been to develop a better understanding of the regulation of ornithine decarboxylase (ODC) during the neoplastic process, and to determine whether induction of ODC is a necessary component in the action of the ras oncogene. Specifically, we have studied the role of ODC overexpression in signaling pathways mediated by Raf or RhoA. Cells transformed by ras are known to have constitutively high levels of ODC activity that correlate with oncogenic transformation. To determine which pathways downstream of Ras contribute to the regulation of ODC activity, NIH-3T3 cells were transfected with plasmids coding for activated mutants of either H-Ras or RhoA, or oncogenic nu-Raf. There was a good correlation between increasing ODC specific activity and change in morphology from normal to transformed in the nu-Raf, HRas(61L), and RhoA(63L) clones. Increasing ODC activity also correlated positively with the ability to grow in soft agar in both the H-Ras- and RhoA-expressing cells. In stable transfections, coexpression of the ODC dominant negative mutant K69A/C360A with either HRas(61L) or RhoA(63L) both inhibited intracellular ODC activity and caused a reversion of the transformed phenotype, as measured by a dramatic reduction in the ability of these cells to grow in soft agar and form foci on a monolayer. These results suggest strongly that ODC induction is necessary for transformation by oncogenic Ras. In contrast, expression of K69A/ C360A had no effect on the ability of nu-Raf-transformed cells to grow in soft agar, although intracellular ODC levels were inhibited. When grown on a monolayer, these cells also maintained their transformed appearance. Furthermore, expression of the ODC dominant negative mutant did not affect the phosphorylation of mitogen-activated protein kinase in nu-Raf-transformed cells. These experiments provide strong support for the concept that transformation by activated ras is accompanied by an induction of ODC. The results using RhoA(63L) and nu-Raf suggest that this increase in ODC activity is mediated at least in part through a Raf/ mitogen-activated protein kinase independent pathway.

3T3 Cells↗

PubMed1998

A new model for how O6-methylguanine-DNA methyltransferase binds DNA.

Human methyltransferase (hAT) catalyzes the transfer of an alkyl group from the 6-position of guanine to an active site Cys residue. The physiological role of hAT is the repair of alkylated guanine residues in DNA. However, the repair of methylated or chloroethylated guanine bases negates the effects of certain chemotherapeutic agents. A model of how hAT binds DNA might be useful in the design of compounds that could inactivate hAT. We have used computer modeling studies to generate such a model. The model utilizes a helix-loop-wing DNA binding motif found in Mu transposase. The model incorporates a flipped out guanine base in order to bring the methylated oxygen atom close to the active site Cys residue. The model is consistent with a variety of chemical and biochemical data.

Amino Acid Sequence↗

PubMed1998

O6-alkylguanine-DNA alkyltransferase inactivation by ester prodrugs of O6-benzylguanine derivatives and their rate of hydrolysis by cellular esterases.

To modulate the bioavailability and perhaps improve the tumor cell selectivity of O6-alkylguanine-DNA alkyltransferase (AGT) inactivators, pivaloyloxymethyl ester derivatives of O6-benzylguanine (BG) were synthesized and tested as AGT inactivators and as substrates for cellular esterases. The potential prodrugs examined were the 7- and 9-pivaloyloxymethyl derivatives of O6-benzylguanine (7- and 9-esterBG), and of 8-aza-O6-benzylguanine (8-aza-7-esterBG and 8-aza-9-esterBG) and the 9-pivaloyloxymethyl derivative of 8-bromo-O6-benzylguanine (8-bromo-9-esterBG). The benzylated purines were all potent inactivators of the pure AGT and of the AGT activity in HT29 cells and cell extracts. Each ester was at least 75 times less potent than the corresponding benzylated purine against the pure human AGT. In contrast, the activities of esters and their respective benzylated purine were similar in crude cell extracts and in intact cells. The increase in potency of esters in cellular extracts could be explained by a conversion of the respective prodrug to the more potent benzylated purine in the presence of cellular esterases. The apparent catalytic activity (Vmax/Km) of liver microsomal esterase for 8-azaBG ester prodrugs was 70-130 times greater than for BG prodrugs and 10-20 times greater than for 8-bromo-9-esterBG. Tumor cell hydrolysis of the esters varied considerably as a function of cell type and prodrug structure. These data suggest that these or related prodrugs may be advantageous for selective AGT inactivation in certain tumor types.

Animals↗

PubMed1998

Reaction of O6-benzylguanine-resistant mutants of human O6-alkylguanine-DNA alkyltransferase with O6-benzylguanine in oligodeoxyribonucleotides.

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.

Amino Acid Sequence↗

PubMed1998

Isolation of human O6-alkylguanine-DNA alkyltransferase mutants highly resistant to inactivation by O6-benzylguanine.

The activity of O6-alkylguanine-DNA alkyltransferase (AGT) protects cells from killing by methylating or chloroethylating agents. AGT is strongly inhibited by O6-benzylguanine (ED50, 0.2 microM), and this drug is presently undergoing clinical trials to enhance chemotherapy by alkylating agents. Point mutations such as P140A (ED50, 5 microM) render AGT resistant to O6-benzylguanine (BG). Selection for such mutants may prove to be a problem in the use of BG, and a better knowledge of the factors underlying resistance to BG will enable the rational design of improved inhibitors able to inactivate these mutants. BG-resistant AGT mutants may also be valuable for expression in bone marrow stem cells to reduce myelosuppression brought about by alkylating agents, to increase the therapeutic index of therapies including BG, and for use as a selectable marker to allow other genes to be expressed in such stem cells. We have therefore set up a general screen to obtain such mutants by using the ability of AGT to protect Escherichia coli GWR109 lacking endogenous AGT from killing by N-methyl-N'-nitro-N-nitrosoguanidine. When the cells were rendered permeable to BG by mutating the lipopolysaccharide membrane component forming strain TRG8, the protection by AGT expression was abolished by treating the cells with BG. The known P140A mutant was used to test the system and was highly selected for by treatment with 50 microM BG and 40 microg/ml N-methyl-N'-nitro-N-nitrosoguanidine. The sequence coding for PVP at positions 138-140 in AGT was replaced with a random nucleotide sequence, and this library was used to transform TRG8. All of the 59 colonies analyzed having AGT activity that survived the selection from the pool of 36,000 transformants were resistant to BG. Many (69%) of these mutants contained lysine at position 140, and all of these showed the highest level of resistance with <10% loss of activity when crude cell extracts were incubated with 1.2 mM BG. This result was confirmed with three mutants (P138K/V139L/P140K, P138M/V139L/P140K, and P140K), which were purified to homogeneity. The next most common residues found at position 140 were arginine (7%) and asparagine (7%). Studies carried out with purified preparations of mutants P140R and P140N revealed that these mutations also provided resistance to BG but to a lesser extent than P140K (ED50s of 190 and 7 microM, respectively). These results indicate that: (a) this screening method can be used to evaluate BG resistance of single or multiple changes throughout the AGT sequence; and (b) replacement of proline-140 with lysine is the most effective point mutation at this site causing BG resistance and is more than 200 times more effective than replacement with alanine.

Colony Count, Microbial↗

PubMed1998

Probing of conformational changes in human O6-alkylguanine-DNA alkyl transferase protein in its alkylated and DNA-bound states by limited proteolysis.

Human O6-alkylguanine-DNA alkyl transferase (hAGT) is a DNA repair protein that protects cells from alkylation damage by transferring an alkyl group from the O6-position of guanine to a cysteine residue in the active site (-PCHR-) of the protein. The structure of the hAGT protein (23 kDa) has been probed by limited proteolysis with trypsin and Glu-C endoproteases and analysis of the polypeptide fragments by SDS/PAGE. The native hAGT protein had limited accessibility to digestion with trypsin and Glu-C in spite of a number of potential cleavage sites. Initial cleavage by trypsin occurred at residue Lys-193 to give a 21 kDa polypeptide fragment, and this polypeptide underwent further cleavage at residues Arg-128 and Lys-165. These trypsin-cleavage sites became more accessible to digestion in the presence of double-stranded DNA (dsDNA), indicating that hAGT undergoes a change in its conformation on binding to DNA. However, the trypsin cutting site at the Arg-128 position was less available for digestion in the presence of single-stranded DNA (ssDNA), suggesting that the hAGT protein has a different conformation when bound to ssDNA compared with dsDNA. When protease digestion was carried out on wild-type protein, preincubated with the low-molecular-mass pseudosubstrate O6-benzylguanine, increased susceptibility to proteases was observed. A mutant C145A hAGT protein, which cannot repair O6-alkylguanine because the Cys-145 acceptor site in the active site of the protein is changed to Ala, showed identical trypsin cleavage to the wild type, but its digestion was not affected by O6-benzylguanine. These results suggest that alkylation of hAGT leads to an altered conformation. The acquisition of increased susceptibility to proteases upon DNA binding and alkylation demonstrates that hAGT undergoes considerable conformational changes in its structure upon binding to DNA and after repair of alkylation damage.

Alkylation↗