Role of codon 160 in the sensitivity of human O6-alkylguanine-DNA alkyltransferase to O6-benzylguanine.

O6-Alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein that provides protection from alkylating agents such as dacarbazine, temozolomide, and 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), which are used for cancer chemotherapy. O6-Benzylguanine (BG) is an inhibitor of AGT that sensitizes tumors to these agents. BG is currently in clinical trials. It is possible that the presence of resistant forms of AGT may limit the effectiveness of this strategy. Previous studies have shown that the AGT mutant G160R, which may occur naturally as a result of a polymorphism in the AGT gene, is resistant to BG, whereas the mutants G160W and G160A are actually more sensitive to the inhibitor. To examine other mutations at this site, a random sequence was placed at codon 160 in the AGT cDNA, and a plasmid library was constructed to express these sequences in Escherichia coli. After selection with BG and N-methyl-N'-nitro-N-nitrosoguanidine, BG-resistant mutants were obtained and analyzed. Eleven different amino acid substitutions were found to impart BG resistance by this assay. The most resistant mutants contained histidine or arginine, which had EC50 values of 12 and 4.7 microM, respectively, compared with the wild-type EC50 of 0.08 microM, but nine other alterations led to at least a 10-fold rise in the EC50 value. Three additional mutations at codon 160 were constructed by site-directed mutagenesis, and these led to 6- to 11-fold increases in resistance to BG. Comparisons of the properties of mutants G160R and G160E showed that the presence of DNA enhanced the reaction with BG much more strongly when an acidic residue was present at this position. This may account for the lack of selection of the G160E mutation even though it did impart resistance to BG. These results indicate that many alterations of AGT at position 160 can lead to significant resistance to BG.     

Effect of alterations of key active site residues in O6-alkylguanine-DNA Alkyltransferase on its ability to modulate the genotoxicity of 1,2-dibromoethane

The production of mutations and the reduction in survival of cells treated with alpha,omega-dihaloalkanes is greatly enhanced by the presence of O6-alkylguanine-DNA alkyltransferase (AGT), a DNA repair protein that removes O6-alkylguanine adducts from DNA [Liu, L., Hachey, D. L., Valadez, G., Williams, K. M., Guengerich, F. P., Loktionova, N. A., Kanugula, S., and Pegg, A. E. (2004) J. Biol. Chem. 279, 4250-4259]. The effects of alterations to key residues in the active site of AGT were studied using AGTs with point mutations. It was found that mutants of AGT at positions Tyr114, Arg128, Pro140, Gly156, Gly160, and Tyr158 did not bring about the increase in genotoxicity of 1,2-dibromoethane seen with wild-type AGT, although these mutants, with the exception of those at Tyr114 and Arg128, are known to have sufficient AGT repair function to be able to protect cells from alkylating agents. The R128A mutant was able to react with 1,2-dibromoethane at the Cys145 acceptor site, but the resulting AGT-Cys145S-(CH2)2Br was much less able to produce a covalent adduct with DNA. This result is explained by the need for AGT to induce a structural change in the DNA "flipping" of a guanine nucleotide into the substrate binding pocket where Cys145 is located since the side chain of residue Arg128 plays a critical role in this reaction. Point mutations in AGT at the other sites (Y114A, P140K, and Y158H) reduced the ability of the protein to react with 1,2-dibromoethane as measured by the loss of activity. These results were confirmed by MS analysis of the tryptic peptide that contains the modified Cys145. There was no change in the stability of the AGT-Cys145S-(CH2)2Br intermediate formed in mutants Y158H and P140K. The reaction was studied in detail with mutant P140K using dihaloalkanes of different length; no effect of the mutations was seen with dibromomethane, but an enhanced difference was observed with 1,3-dibromopropane and 1,5-dibromopentane. These results show that even slight alterations in the active site pocket of AGT that do not prevent its ability to protect cells from alkylating agents can block the paradoxical enhancement of the genotoxicity of the larger alpha,omega-dihaloalkanes by reducing the reaction with Cys145.     

The repair of the tobacco specific nitrosamine derived adduct O6-[4-Oxo-4-(3-pyridyl)butyl]guanine by O6-alkylguanine-DNA alkyltransferase variants

The tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent pulmonary carcinogen, both methylates and pyridyloxobutylates DNA. Both reaction pathways generate promutagenic O6-alkylguanine adducts. These adducts, O6-methylguanine (O6-mG) and O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobG), are repaired by O6-alkylguanine-DNA alkyltransferase (AGT). In this report, we demonstrate that pyridyloxobutyl DNA adducts are repaired by AGT in a reaction that results in pyridyloxobutyl transfer to the active site cysteine. Because minor changes within the binding pocket of AGT can alter the ability of this protein to repair bulky O6-alkylguanine adducts relative to O6-mG, we explored the ability of AGTs from different species as well as several human AGT variants and mutants to discriminate between O6-mG or O6-pobG adducts. We incubated proteins with equal molar amounts of oligodeoxynucleotides containing site specifically incorporated O6-mG or O6-pobG and measured repair. Bacterial AGTs poorly repaired O6-pobG. Mouse and rat AGT repaired both adducts at comparable rates. Wild-type human AGT, variant I143V/K178R, and mutant N157H repaired O6-mG approximately twice as fast as O6-pobG. Human variant G160R and mutants P140K, Y158H, G156A, and E166G did not repair O6-pobG until all of the O6-mG was removed. To understand the role of adduct structure on relative repair rates, the competition experiments were repeated with two other bulky O6-alkylguanine adducts, O6-butylguanine (O6-buG) and O6-benzylguanine (O6-bzG). The proteins displayed similar repair preference of O6-mG relative to O6-buG as observed with O6-pobG. In contrast, all of the mammalian proteins, except the mutant P140K, preferentially repaired O6-bzG. These studies indicate that the rate of repair of O6-pobG is highly dependent on protein structure. Inefficient repair of O6-pobG by bacterial AGT explains the high mutagenic activity of this adduct in bacterial systems. In addition, differences observed in the repair of this adduct by mammalian proteins may translate into differences in sensitivity to the mutagenic and carcinogenic effects of NNK or other pyridyloxobutylating nitrosamines.     

Interaction of mammalian O(6)-alkylguanine-DNA alkyltransferases with O(6)-benzylguanine

Human O(6)-alkylguanine-DNA alkyltransferase (hAGT) activity is a major factor in providing resistance to cancer chemotherapeutic alkylating agents. Inactivation of hAGT by O(6)-benzylguanine (BG) is a promising strategy for overcoming this resistance. Previous studies, which have focused on the region encompassed by residues Pro138 to Gly173, have identified more than 100 individual mutations located at 23 discrete sites at which alterations can render AGT less sensitive to BG. We have now extended the examination of possible sites in hAGT at which alterations might lead to BG resistance to include the residues from Val130 to Asn137, which also make up part of the binding pocket into which BG is postulated to fit. A further 21 mutations located at positions Gly132, Met134, Arg135, and Gly136 were found to lower sensitivity to BG. Mutants R135L, R135Y, and G136P were the most strikingly resistant, with a 50-fold increase in the amount of BG needed to obtain 50% inactivation. These results therefore increase the number of sites at which BG resistance can occur in response to a single amino acid change to 27. Although mammalian AGTs are very similar in amino acid sequence, mouse AGT (mAGT) is significantly less sensitive to BG than rat AGT (rAGT) or hAGT. Construction of chimeric proteins in which portions came from the rAGT and the mAGT indicated that the difference in inactivation resided solely in the amino acids located in the sequence from residues 150 to 188. Individual mutations of the three residues where rAGT and mAGT differ in this region showed that the principal reason for the reduced ability of the mAGT to react with BG was the presence of a histidine residue at position 161, which is occupied by asparagine in rAGT and hAGT. These experiments indicate that many minor changes in amino acids forming all parts of the nucleoside binding pocket of AGT can alter its ability to react with BG and that the possibility that polymorphisms or variants may occur reducing the effectiveness of combination therapy with BG and alkylating agents must be considered.     

O~6-苄基鸟嘌呤增强 1,3-二 (2 -氯乙基)-亚硝基脲对人肝癌细胞及其移植瘤的抗肿瘤作用(英文)

应用噻唑蓝 (MTT)法检测 O6-苄基鸟嘌呤(O6- BG)与 1 ,3-二 (2 -氯乙基 ) -亚硝基脲 (BCNU)合用的细胞毒作用及透射电镜检测凋亡细胞的方法研究了 O6- BG对 O6-烷基鸟嘌呤 - DNA烷基转移酶(O6- AGT )阳性的人肝癌细胞 SMMC- 772 1对BCNU细胞毒作用敏感性的影响及其与 BCNU合用治疗移植瘤的协同效果 .结果显示 :1 .5- 6.0 mg· L-1的 O6- BG预先作用 2 h后 ,SMMC- 772 1细胞对 BCNU的敏感性明显增加 ;0 .75- 6.0 mg· L-1的 O6- BG可完全快速地抑制肿瘤细胞的 AGT活性并持续 1 2 h;ip 90 mg· kg-1的 O6- BG预处理 2 h后给予 2 5mg·kg-1的 BCNU治疗 ,可使动物 sc接种的人肝癌移植瘤生长延迟 38.6d,诱导肿瘤细胞凋亡 ,并且可明显抑制肿瘤组织的转移酶活性 .说明 O6- BG与 BCNU合用于 AGT阳性的肿瘤将具有明显的治疗效果     

Random mutagenesis of the human adenosine A2B receptor followed by growth selection in yeast. Identification of constitutively active and gain of function mutations

To gain insight in spontaneous as well as agonist-induced activation of the human adenosine A2B receptor, we applied a random mutagenesis approach in yeast to create a large number of receptor mutants and selected mutants of interest with a robust screening assay based on growth. The amino acid sequence of 14 mutated receptors was determined. All these mutated receptors displayed constitutive activity. In particular, single-point mutations at T42A, V54L, and F84S and a triple-point mutation at N36S, T42A, and T66A resulted in high constitutive activity. In addition, a C-terminally truncated (after Lys269) mutant, Q214L I230N V240M V250M N254Y T257S K269stop, was highly constitutively active. The T42A, V54L, and F84S mutants showed a considerable decrease, 4.9- to 6.9-fold, in the EC50 value of 5'-N-ethylcarboxamidoadenosine (NECA), an adenosine analog. Combined mutation of I242T, K269R, V284A, and H302Q, as well as F84L together with S95G, resulted in an even greater potency of NECA of 10- and 18-fold, respectively. In fact, all constitutively active mutants had an increased potency for NECA. This suggests that the wild-type (wt) human A2B receptor itself is rather silent, which may explain the low affinity of agonists for this receptor. To verify the ability of the mutant receptors to activate mammalian second messenger systems, cAMP experiments were performed in CHO cells stably expressing the wt and T42A receptors. These experiments confirmed the increased sensitivity of T42A for NECA, because the EC50 values of T42A and the wt receptor were 0.15 +/- 0.04 and 1.3 +/- 0.4 microM, respectively.     

Involvement of basic amino acid residues in transmembrane regions 6 and 7 in agonist and antagonist recognition of the human platelet P2Y(12)-receptor

The P2Y(12)-receptor plays a prominent role in ADP-induced platelet aggregation. In the present study, we searched for amino acid residues involved in ligand recognition of the human P2Y(12)-receptor. Wild-type or mutated receptors were expressed in 1321N1 astrocytoma cells and Chinese hamster ovary (CHO) cells. There were no major differences in cellular expression of the constructs. Cellular cAMP production and cAMP response element (CRE)-dependent luciferase expression was increased by isoproterenol (astrocytoma cells) or forskolin (CHO cells). In cells expressing wild-type receptors, R256K or S101A mutant constructs, 2-methylthio-ADP inhibited the induced cAMP production with IC(50) concentrations of about 0.3nM. In cells expressing R256A constructs, the IC(50) concentration amounted to 25nM. In cells expressing H253A/R256A, Y259D and K280A constructs, 2-methylthio-ADP failed to affect the cellular cAMP production. Moreover, in cells expressing Y259D and K280A constructs, 2-methylthio-ADP did also not change the forskolin-induced CRE-dependent luciferase expression and caused only small increases in the serum response element-dependent luciferase expression. The antagonist cangrelor had similar potencies at wild-type receptors and R256A constructs (apparent pK(B)-value at wild-type receptors: 9.2). In contrast, reactive blue-2 had a lower potency at the R256A construct (apparent pK(B)-value at wild-type receptors: 7.6). In summary, the data indicate the involvement of Arg256, Tyr259 and, possibly, H253 (transmembrane region TM6) as well as Lys280 (TM7) in the function of the human P2Y(12)-receptor. Arg256 appears to play a role in the recognition of nucleotide agonists and the non-nucleotide antagonist reactive blue-2, but no role in the recognition of the nucleotide antagonist cangrelor.     

Non-synonymous single nucleotide alterations in the microsomal epoxide hydrolase gene and their functional effects

1. By sequencing genomic DNA from 72 established cell lines derived from Japanese individuals, we detected 25 single nucleotide alterations in the microsomal epoxide hydrolase (EPHX1) gene. Of them, five were exonic alterations resulting in amino acid alterations (77C>G, T26S; 128G>C, R43T; 337T>C, Y113H; 416A>G, H139R; 823A>G, T275A). The T26S, R43T, Y113H and H139R substitutions were found at relatively high frequencies and seemed to be polymorphic, and T26S and T275A were novel. 2. To examine the effects of these amino acid alterations on EPHX1 function, EPHX1 cDNA constructs of wild-type and five variants were transfected into COS-1 cells, and their hydrolytic activities for cis -stilbene oxide were determined in vitro. Although all of the transfectants expressed EPHX1 mRNA and protein at similar levels, the variant H139R protein was expressed at a significantly higher level (128% of the wild-type). K m values were not significantly different between the wild-type and variants. 3. Increase (140%) in the enzymatic activity (V max) of the variant H139R was accompanied by the increased EPHX1 protein level without any significant change in the intrinsic EPHX1 activity. On the other hand, the variant R43T showed increased values for V max and clearance (V max / K m) (around 130%) both on a microsomal protein basis and on a EPHX1 protein basis. 4. These results suggest that R43T as well as H139R increase epoxide hydrolase activity.     

Non-synonymous single nucleotide alterations in the microsomal epoxide hydrolase gene and their functional effects

1. By sequencing genomic DNA from 72 established cell lines derived from Japanese individuals, we detected 25 single nucleotide alterations in the microsomal epoxide hydrolase (EPHX1) gene. Of them, five were exonic alterations resulting in amino acid alterations (77C>G, T26S; 128G>C, R43T; 337T>C, Y113H; 416A>G, H139R; 823A>G, T275A). The T26S, R43T, Y113H and H139R substitutions were found at relatively high frequencies and seemed to be polymorphic, and T26S and T275A were novel. 2. To examine the effects of these amino acid alterations on EPHX1 function, EPHX1 cDNA constructs of wild-type and five variants were transfected into COS-1 cells, and their hydrolytic activities for cis-stilbene oxide were determined in vitro. Although all of the transfectants expressed EPHX1 mRNA and protein at similar levels, the variant H139R protein was expressed at a significantly higher level (128% of the wild-type). K(m) values were not significantly different between the wild-type and variants. 3. Increase (140%) in the enzymatic activity (V(max)) of the variant H139R was accompanied by the increased EPHX1 protein level without any significant change in the intrinsic EPHX1 activity. On the other hand, the variant R43T showed increased values for V(max) and clearance (V(max)/K(m)) (around 130%) both on a microsomal protein basis and on a EPHX1 protein basis. 4. These results suggest that R43T as well as H139R increase epoxide hydrolase activity.     

O6-苄基鸟嘌呤增强1，3-二（2-氯乙基）-亚硝基脲对人肝癌细胞及其移植瘤的抗肿瘤作用

应用噻唑蓝（MTT）法检测O⁶-苄基鸟嘌呤(O⁶-BG)与1,3-二（2-氯乙基）-亚硝基脲（BCNU）合用的细胞毒作用及透射电镜检测凋亡细胞的方法研究了O⁶-BG对O⁶-烷基鸟嘌呤-DNA烷基转移酶（O⁶-AGT）阳性的人肝癌细胞SMMC-7721对BCNU细胞毒作用敏感性的影响及其与BCNU合用治疗移植瘤的协同效果. 结果显示：1.5-6.0 mg·L^-1的O⁶-BG预先作用2 h后,SMMC- 7721细胞对BCNU的敏感性明显增加;0.75-6.0 mg·L^-1的O⁶-BG可完全快速地抑制肿瘤细胞的AGT活性并持续12 h; ip 90 mg·kg^-1的O⁶-BG预处理2 h后给予25 mg·kg^-1的BCNU治疗,可使动物sc接种的人肝癌移植瘤生长延迟38.6 d, 诱导肿瘤细胞凋亡,并且可明显抑制肿瘤组织的转移酶活性. 说明O⁶-BG与BCNU合用于AGT阳性的肿瘤将具有明显的治疗效果.     

O6-alkylguanine-DNA alkyltransferase has opposing effects in modulating the genotoxicity of dibromomethane and bromomethyl acetate

O(6)-Alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein that removes O(6)-alkylguanine adducts. The interaction of dibromomethane (CH(2)Br(2)) and bromomethyl acetate (BrCH(2)OAc) with AGT was studied in vitro, and the effect of AGT on their toxicity and mutagenicity was investigated using Escherichia coli strain TRG8 (lacking endogenous AGT) that expressed human AGT or its inactive C145A mutant. Both CH(2)Br(2) and BrCH(2)OAc reacted with AGT at its cysteine acceptor site, abolishing its DNA repair activity with the latter agent being much more potent. The formation of AGT-Cys(145)S-CH(2)OAc by BrCH(2)OAc was confirmed by mass spectral analysis, but the presumed AGT-Cys(145)S-CH(2)Br adduct from CH(2)Br(2) was too unstable for such characterization. In the presence of CH(2)Br(2), AGT was covalently cross-linked to an oligodeoxyribonucleotide, 5'-d(AG)(8)-3', but no cross-link was formed by BrCH(2)OAc. Survival of cells exposed to CH(2)Br(2) was reduced, and the number of mutants was greatly increased when wild-type AGT was present. The cytotoxicity of CH(2)Br(2) was similar to that of BrCH(2)CH(2)Br(2), but the mutagenicity was about four times less. Virtually all of the AGT-mediated mutants induced by CH(2)Br(2) in the rpoB gene were at G:C sites with equal numbers of transitions to A:T and transversions to T:A. In contrast, BrCH(2)OAc was more than 10-fold less genotoxic than CH(2)Br(2) and the survival of cells exposed to BrCH(2)OAc was not affected by AGT. The number of mutations (almost all G:C to A:T transitions) induced by BrCH(2)OAc was slightly reduced by the presence of wild-type AGT and substantially increased by the inactive C145A mutant. These results with CH(2)Br(2) are consistent with a mechanism in which reaction at the active site Cys145 residue followed by attack of AGT-Cys(145)S-CH(2)Br at guanine in DNA forms a covalent adduct, which leads to cytotoxicity and to mutagenicity. The results with BrCH(2)OAc suggest that it reacts directly with DNA to form O(6)-(CH(2)OAc)guanine, which, if unrepaired, causes G:C to A:T transitions. Our experiments reveal two novel pathways (direct inactivation of AGT and formation of AGT-Cys(145)S-CH(2)-DNA adducts) by which CH(2)Br(2) may cause damage to the genome in addition to the well-recognized pathway involving activation by GSTs.     

Assessment of arginine 97 and lysine 72 as determinants of substrate specificity in cytochrome P450 2C9 (CYP2C9).

CYP2C9 is distinguished by a preference for substrates bearing a negative charge at physiological pH. Previous studies have suggested that CYP2C9 residues R97 and K72 may play roles in determining preference for anionic substrates by interaction at the active site or in the access channel. The aim of the present study was to assess the role of these two residues in determining substrate selectivity. R97 and K72 were substituted with negative, uncharged polar and hydrophobic residues using a degenerate polymerase chain reaction-directed strategy. Wild-type and mutant enzymes were expressed in bicistronic format with human cytochrome P450 reductase in Escherichia coli. Mutation of R97 led to a loss of holoenzyme expression for R97A, R97V, R97L, R97T, and R97E mutants. Low levels of hemoprotein were detected for R97Q, R97K, R97I, and R97P mutants. Significant apoenzyme was observed, suggesting that heme insertion or protein stability was compromised in R97 mutants. These observations are consistent with a structural role for R97 in addition to any role in substrate binding. By contrast, all K72 mutants examined (K72E, K72Q, K72V, and K72L) could be expressed as hemoprotein at levels comparable to wild-type. Type I binding spectra were obtained with wild-type and K72 mutants using diclofenac and ibuprofen. Mutation of K72 had little or no effect on the interaction with these substrates, arguing against a critical role in determining substrate specificity. Thus, neither residue appears to play a role in determining substrate specificity, but a structural role for R97 can be proposed consistent with recently published crystallographic data for CYP2C9 and CYP2C5.     

Kinetics of O(6)-methyl-2'-deoxyguanosine repair by O(6)-alkylguanine DNA alkyltransferase within K-ras gene-derived DNA sequences

O(6)-Methyl-2'-deoxyguanosine (O(6)-Me-dG) is a potent mutagenic DNA adduct that can be induced by a variety of methylating agents, including tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). O(6)-Me-dG is directly repaired by the specialized DNA repair protein, O(6)-alkylguanine DNA alkyltransferase (AGT), which transfers the O(6)-alkyl group from the modified guanine to a cysteine thiol within the active site of the protein. Previous investigations suggested that AGT repair of O(6)-alkylguanines may be sequence-dependent as a result of flanking nucleobase effects on DNA conformation and energetics. In the present work, a novel high-performance/pressure liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI+-MS/MS)-based approach was developed to analyze the kinetics of AGT-mediated repair of O(6)-Me-dG adducts placed at different sites within the double-stranded DNA sequence representing codons 8-17 of the K-ras protooncogene, 5'-G1TA G2TT G3G4A G5CT G6G7T G8G9C G10TA G11G12C AAG13 AG14T-3', where G5, G6, G7, G8, G9, G10, or G11 was replaced with O(6)-Me-dG. The second guanine of K-ras codon 12 (G7 in our numbering system) is a major mutational hotspot for G --> A transitions observed in lung tumors of smokers and in neoplasms induced in laboratory animals by exposure to methylating agents. O(6)-Me-dG-containing duplexes were incubated with human recombinant AGT protein, and the reactions were quenched at specific times. Following acid hydrolysis to release purines, isotope dilution HPLC-ESI-MS/MS was used to determine the amounts of O(6)-Me-G remaining in DNA. The relative extent of demethylation for O(6)-Me-dG adducts located at G5, G6, G7, G8, G9, G10, or G11 following a 10 s incubation with AGT showed little variation as a function of sequence position. Furthermore, the second-order rate constants for the repair of O(6)-Me-dG adducts located at the first and second positions of the K-ras codon 12 (5'-G6G7T-3') were similar (1.4 x 10(7) M(-1) s(-1) vs 7.4 x 10(6) M(-1) s(-1), respectively), suggesting that O(6)-Me-dG repair by AGT is not the determining factor for K-ras codon 12 mutagenesis following exposure to methylating agents. The new HPLC-ESI-MS/MS assay developed in this work is a valuable tool which will be used to further explore the role of local sequence environment and endogenous DNA modifications in shaping mutational spectra of NNK and other methylating agents.     

Improvement of cyclophosphamide activation by CYP2B6 mutants: from in silico to ex vivo

Cyclophosphamide (CPA) is a chemotherapeutic agent that is primarily activated in the liver by cytochrome P4502B6 (CYP2B6) and then transported to the tumor via blood flow. To prevent deleterious secondary effects, P450-based gene-directed enzyme prodrug therapy (GDEPT) consists of expressing CYP2B6 in tumor cells before CPA treatment. Given the relatively low affinity of CYP2B6 for CPA, the aim of our work was to modify CYP2B6 to increase its catalytic efficiency (V(max)/K(m)) to metabolize CPA into 4'-OH CPA. A molecular model of CYP2B6 was built, and four residues in close contact with the substrate were subjected to mutagenesis. Canine CYP2B11 exhibiting a particularly low K(m) to CPA, the amino acids exclusively present in the CYP2B11 substrate recognition sequences were substituted in human CYP2B6. All mutants (n = 26) were expressed in Saccharomyces cerevisiae and their enzymatic constants (K(m), V(max)) evaluated using CPA as substrate. Five mutants exhibited a 2- to 3-fold higher catalytic efficiency than wild-type CYP2B6. A double mutant, comprising the two most effective mutations, showed a 4-fold increase in K(m)/V(max). Molecular dynamic simulations of several mutants were found to be consistent with the observed modifications in catalytic efficiency. Finally, expression of the CYP2B6 114V/477W double mutant, contrary to wt CYP2B6, allowed switching of a resistant human head and neck cancer cell line (A-253) into a sensitive cell line toward CPA. Thus, we were able to obtain a new efficient CYP2B6 mutant able to metabolize CPA, an important step in the GDEPT strategy for human cancer treatment.     

Non-synonymous single nucleotide alterations found in the CYP2C8 gene result in reduced in vitro paclitaxel metabolism.

By sequencing genomic DNA from 73 established cell lines derived from Japanese individuals, we detected 9 single nucleotide polymorphisms (SNPs) in the CYP2C8 gene. Of them, 3 exonic SNPs resulted in amino acid alterations (g416a, R139K; a1196g, K399R; c1210g, P404A). The first two alterations were detected concurrently in one cell line and thought to be the same as CYP2C8*3. To examine the effects of these amino acid alterations on CYP2C8 function, wild-type and four types of variant CYP2C8 cDNA constructs (R139K, K399R, R139K/K399R and P404A) were transfected into Hep G2 cells and their paclitaxel 6a-hydroxylase activities were determined in vitro. Km values were not significantly different from that of the wild-type in any of the variants studied. The variant R139K/K399R showed reduced values for Vmax and clearance (Vmax/Km) similar to those of its single variant, R139K. The variant P404A also showed a significantly lowered clearance due to reduced level of protein expression. These results suggest that not only the double variant (R139K/K399R, CYP2C8*3) but also our novel variant P404A in the CYP2C8 gene are less efficient in paclitaxel metabolism.     

Debenzylation of O(6)-benzyl-8-oxoguanine in human liver: implications for O(6)-benzylguanine metabolism

O(6)-Benzylguanine (BG) effectively inactivates the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase, and enhances the effectiveness of 1,3-bis(2-chloroethyl)-1-nitrosourea in cells in culture and tumor-bearing animals. BG is presently in phase II clinical trials. In humans, BG is converted to O(6)-benzyl-8-oxoguanine (8-oxoBG), a longer-lived, yet equally potent inactivator. We have isolated and identified the debenzylated product, 8-oxoguanine, in plasma and urine of patients following administration of BG. The purpose of this work was to determine the human liver enzymes responsible for the debenzylation of 8-oxoBG. Therefore, 8-oxoBG was incubated with human liver microsomes and cytosol, and the concentration of 8-oxoguanine was determined. No appreciable product was formed in the cytosol; however, increasing amounts of 8-oxoguanine were formed with increasing concentrations of pooled human liver microsomes. The amount of 8-oxoguanine formed increased with time and substrate concentration. Co-incubation of human liver microsomes with 8-oxoBG and various cytochrome P450 isoform-selective inhibitors suggested the possible involvement of CYP1A2, 2E1, and/or 2A6 in this reaction. Incubation of 8-oxoBG with baculovirus cDNA-overexpressed CYP1A2, 2E1, 2A6, and 3A4 demonstrated that formation of 8-oxoguanine was due mainly to CYP1A2. Debenzylation of 8-oxoBG complied with Michaelis-Menten kinetics with K(m) and V(max) values of 35.9 microM and 0.59 pmol/min/pmol of CYP1A2, respectively. CYP1A2 appears to be mainly responsible for the debenzylation of 8-oxoBG in human liver.     

KS900: A hypoxia-directed, reductively activated methylating antitumor prodrug that selectively ablates O(6)-alkylguanine-DNA alkyltransferase in neoplastic cells

To most effectively treat cancer it may be necessary to preferentially destroy tumor tissue while sparing normal tissues. One strategy to accomplish this is to selectively cripple the involved tumor resistance mechanisms, thereby allowing the affected anticancer drugs to gain therapeutic efficacy. Such an approach is exemplified by our design and synthesis of the intracellular hypoxic cell activated methylating agent, 1,2-bis(methylsulfonyl)-1-methyl-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS900) that targets the O-6 position of guanine in DNA. KS900 is markedly more cytotoxic in clonogenic experiments under conditions of oxygen deficiency than the non-intracellularly activated agents KS90, and 90M, when tested in O(6)-alkylguanine-DNA alkyltransferase (AGT) non-expressing cells (EMT6 mouse mammary carcinoma, CHO/AA8 hamster ovary, and U251 human glioma), and than temozolomide when tested in AGT expressing cells (DU145 human prostate carcinoma). Furthermore, KS900 more efficiently ablates AGT in HL-60 human leukemia and DU145 cells than the spontaneous globally activated methylating agent KS90, with an IC(50) value over 9-fold lower than KS90. Finally, KS900 under oxygen-deficient conditions selectively sensitizes DU145 cells to the chloroethylating agent, onrigin, through the ablation of the resistance protein AGT. Thus, under hypoxia, KS900 is more cytotoxic at substantially lower concentrations than methylating agents such as temozolomide that are not preferentially activated in neoplastic cells by intracellular reductase catalysts. The necessity for intracellular activation of KS900 permits substantially greater cytotoxic activity against cells containing the resistance protein O(6)-alkylguanine-DNA alkyltransferase (AGT) than agents such as temozolomide. Furthermore, the hypoxia-directed intracellular activation of KS900 allows it to preferentially ablate AGT pools under the oxygen-deficient conditions that are present in malignant tissue.     

Resistance-modifying agents. 8. Inhibition of O(6)-alkylguanine-DNA alkyltransferase by O(6)-alkenyl-, O(6)-cycloalkenyl-, and O(6)-(2-oxoalkyl)guanines and potentiation of temozolomide cytotoxicity in vitro by O(6)-(1-cyclopentenylmethyl)guanine

A series of O(6)-allyl- and O(6)-(2-oxoalkyl)guanines were synthesized and evaluated, in comparison with the corresponding O(6)-alkylguanines, as potential inhibitors of the DNA-repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT). Simple O(6)-alkyl- and O(6)-cycloalkylguanines were weak AGT inactivators compared with O(6)-allylguanine (IC(50) = 8.5 +/- 0.6 microM) with IC(50) values ranging from 100 to 1000 microM. The introduction of substituents at C-2 of the allyl group of O(6)-allylguanine reduced activity compared with the parent compound, while analogous compounds in the O(6)-(2-oxoalkyl)guanine series exhibited very poor activity (150-1000 microM). O(6)-Cycloalkenylguanines proved to be excellent AGT inactivators, with 1-cyclobutenylmethylguanine (IC(50) = 0.55 +/- 0.02 microM) and 1-cyclopentenylmethylguanine (IC(50) = 0.39 +/- 0.04 microM) exhibiting potency approaching that of the benchmark AGT inhibitor O(6)-benzylguanine (IC(50) = 0.18 +/- 0.02 microM). 1-Cyclopentenylmethylguanine also inactivated AGT in intact HT29 human colorectal carcinoma cells (IC(50) = 0.20 +/- 0.07 microM) and potentiated the cytotoxicity of the monomethylating antitumor agent Temozolomide by approximately 3- and 10-fold, respectively, in the HT29 and Colo205 tumor cell lines. The observation that four mutant AGT enzymes resistant to O(6)-benzylguanine also proved strongly cross-resistant to 1-cyclopentenylmethylguanine indicates that the O(6)-substituent of each compound makes similar binding interactions within the active site of AGT.