Point mutations at multiple sites including highly conserved amino acids maintain activity, but render O6-alkylguanine–DNA alkyltransferase insensitive to O6-benzylguanine

2000 ◽  
Vol 347 (2) ◽  
pp. 519-526
Author(s):  
Meng XU-WELLIVER ◽  
Anthony E. PEGG
Keyword(s):  
Alkylating Agents ◽  
Screening Method ◽  
Point Mutations ◽  
Binding Pocket ◽  
Tumour Cells ◽  
Repair Protein ◽  
Dna Repair Protein ◽  
Dna Alkyltransferase ◽  
Resistant Mutants ◽  
Conserved Amino Acids

The DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), is inactivated by reaction with the pseudosubstrate, O6-benzylguanine (BG). This inactivation sensitizes tumour cells to chemotherapeutic alkylating agents, and BG is aimed at enhancing cancer treatment in clinical trials. Point mutations in a 24 amino acid sequence likely to form the BG-binding pocket were identified using a screening method designed to identify BG-resistant mutants. It was found that alterations in 21 of these residues were able to render AGT resistant to BG. These included mutations at the highly conserved residues Lys165, Leu168 and Leu169. The two positions at which changes led to the largest increase in resistance to BG were Gly156 and Lys165. Eleven mutants at Gly156 were identified, with increases in resistance ranging from 190-fold (G156V) to 4400-fold (G156P). Two mutants at Lys165 found in the screen (K165S and K165A) showed 620-fold and 100-fold increases in resistance to BG. Two mutants at the Ser159 position (S159I and S159V) were > 80-fold more resistant than wild-type AGT. Eleven active mutants at Leu169 were also resistant to BG, but with lower increases (5-86-fold). Fourteen BG-resistant mutants were found for position Cys150, with 3-26-fold increases in the amount of inhibitor needed to produce a 50% loss of activity in a 30 min incubation. Six BG-resistant mutants at Asn157 were found with increases of 4-13-fold. These results show that many changes can render human AGT resistant to BG without preventing the ability to protect tumour cells from therapeutic alkylating agents.

scholarly journals Development of a Novel Fluorescence Assay Based on the Use of the Thrombin-Binding Aptamer for the Detection ofO6-Alkylguanine-DNA Alkyltransferase Activity

2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Maria Tintoré ◽  
Anna Aviñó ◽  
Federico M. Ruiz ◽  
Ramón Eritja ◽  
Carme Fàbrega
Keyword(s):  
Conformational Changes ◽  
Alkylating Agents ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Methyl Group ◽  
Quadruplex Structure ◽  
Repair Protein ◽  
Close Proximity ◽  
Dna Repair Protein ◽  
Dna Alkyltransferase

HumanO6-alkylguanine-DNA alkyltransferase (hAGT) is a DNA repair protein that reverses the effects of alkylating agents by removing DNA adducts from theO6position of guanine. Here, we developed a real-time fluorescence hAGT activity assay that is based on the detection of conformational changes of the thrombin-binding aptamer (TBA). The quadruplex structure of TBA is disrupted when a central guanine is replaced by anO6-methyl-guanine. The sequence also contains a fluorophore (fluorescein) and a quencher (dabsyl) attached to the opposite ends. In the unfolded structure, the fluorophore and the quencher are separated. When hAGT removes the methyl group from the central guanine of TBA, it folds back immediately into its quadruplex structure. Consequently, the fluorophore and the quencher come into close proximity, thereby resulting in decreased fluorescence intensity. Here, we developed a new method to quantify the hAGT without using radioactivity. This new fluorescence resonance energy transfer assay has been designed to detect the conformational change of TBA that is induced by the removal of theO6-methyl group.

Conserved residue lysine165 is essential for the ability of O6-alkylguanine–DNA alkyltransferase to react with O6-benzylguanine

2000 ◽  
Vol 347 (2) ◽  
pp. 527-534
Author(s):  
Meng XU-WELLIVER ◽  
Sreenivas KANUGULA ◽  
Natalia A. LOKTIONOVA ◽  
Tina M. CRONE ◽  
Anthony E. PEGG
Keyword(s):  
Dna Repair ◽  
Rate Constant ◽  
Alkylating Agents ◽  
Methylated Dna ◽  
Cell Extracts ◽  
Repair Protein ◽  
Dna Repair Protein ◽  
Dna Alkyltransferase ◽  
Dna Substrates ◽  
Conserved Residue

The role of lysine165 in the activity of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), and the ability of AGT to react with the pseudosubstrate inhibitor, O6-benzylguanine (BG), was investigated by changing this lysine to all other 19 possibilities. All of these mutants (except for K165T, which could not be tested as it was too poorly active for assay in crude cell extracts) gave BG-resistant AGTs with increases in the amount of inhibitor needed to produce a 50% loss of activity in a 30 min incubation (ED50) from 100-fold (K165A) to 2400-fold (K165F). Lys165 is a completely conserved residue in AGTs from many species, and all of the mutations at this site also reduced the ability to repair methylated DNA. The least deleterious change was that to arginine, which reduced the rate constant for DNA repair by approx. 2.5-fold. Mutant K165R resembled all of the other mutants in being highly resistant to BG, with an ED50 value for inactivation by BG > 200-fold greater than wild-type. Detailed studies of purified K165A AGT showed that the rate constant for repair and the binding to methylated DNA substrates were reduced by 10-20-fold. Despite this, the K165A mutant AGT was able to protect cells from alkylating agents and this protection was not abolished by BG. These results show that, firstly, lysine at position 165 is needed for optimal activity of AGT towards methylated DNA substrates and is essential for efficient reaction with BG; and second, even if the AGT activity towards methylated DNA substrates is impaired by mutations at codon 165, such mutants can protect tumour cells from therapeutic alkylating agents. These results raise the possibility that the conservation of Lys165 is due to the need for AGT activity towards substrates containing more bulky adducts than O6-methylguanine. They also suggest that alterations at Lys165 may occur during chemotherapy with BG and alkylating agents and could limit the effectiveness of this therapy.

scholarly journals Modulation of nitrosourea resistance in myeloid leukemias

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1487-1494 ◽  
Author(s):  
SL Gerson ◽  
JE Trey
Keyword(s):  
Dna Repair ◽  
Therapeutic Index ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Biochemical Modulation ◽  
Repair Protein ◽  
Myeloid Leukemias ◽  
Dna Repair Protein ◽  
Dna Alkyltransferase

Abstract Drug resistance in myeloid leukemias may be mediated by an increased capacity to repair chemotherapy-induced DNA damage. Some tumor cell lines that are resistant to nitrosoureas contain the DNA repair protein O6-alkylguanine-DNA alkyltransferase (alkyltransferase). This protects cells by removing cytotoxic, nitrosourea-induced O6-alkylguanine adducts. We measured the level of alkyltransferase activity in myeloid leukemic cells freshly obtained from patients to determine whether the alkyltransferase was an important factor in nitrosourea resistance in these cells and whether inactivation of this protein could sensitize leukemic cells to nitrosoureas. Myeloid leukemic cells from patients with acute nonlymphocytic leukemia and chronic myelogenous leukemia had higher levels of alkyltransferase than did myeloid precursors from normal donors (P less than .01). This difference did not appear to be due to the state of differentiation of the leukemic or normal cells. To show that this repair protein mediated nitrosourea resistance in leukemic cells, cells were treated with the modified base O6- methylguanine to selectively and irreversibly inactivate the alkyltransferase and then exposed to 1,3-bis (2-chloroethyl)-1- nitrosourea (BCNU). An 18-hour incubation in 0.5 mmol/L O6- methylguanine caused an 87% +/- 3.6% decrease in alkyltransferase activity in leukemic cells and a 73% +/- 8.6% decrease in normal myeloid precursors. After treatment with O6-methylguanine, clonogenic leukemic cells from ten different donors became much more sensitive to BCNU, with a decrease in the dose needed to reduce colony survival by 50% (LD50) of 6.3 +/- 1.4-fold. A lesser effect was seen on CFU-GM, BFU- E, and CFU-GEM where the LD50 decreased two- to threefold. These studies show that nitrosourea resistance in myeloid leukemic cells can be abrogated by inactivation of the DNA repair protein O6-alkylguanine- DNA alkyltransferase. This method of biochemical modulation of DNA repair will sensitize leukemic cells to nitrosoureas in vitro and has the potential of increasing the therapeutic index of nitrosoureas in this disease.

scholarly journals Human CD34+ hematopoietic progenitors have low, cytokine-unresponsive O6-alkylguanine-DNA alkyltransferase and are sensitive to O6- benzylguanine plus BCNU

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1649-1655 ◽  
Author(s):  
SL Gerson ◽  
W Phillips ◽  
M Kastan ◽  
LL Dumenco ◽  
C Donovan
Keyword(s):  
Cell Types ◽  
Human Cd34 ◽  
Repair Protein ◽  
Cd34 Cells ◽  
Modification Factor ◽  
Dna Repair Protein ◽  
Dna Alkyltransferase ◽  
Macrophage Colony Stimulating Factor ◽  
Low Levels ◽  
Macrophage Colony

Abstract Human bone marrow (BM) cells contain low levels of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase, which may explain their susceptibility to nitrosourea-induced cytotoxicity and the development of secondary leukemia after nitrosourea treatment. Isolated CD34+ myeloid progenitors were also found to have low levels of alkyltransferase activity. The level of alkyltransferase in CD34+ cells or in mononuclear BM cells did not increase after incubation with granulocyte-macrophage colony-stimulating factor, interleukin-3, stem cell factor, the combination, or 5637 conditioned medium. BCNU sensitivity remained unchanged as well. In addition, O6-benzylguanine depleted alkyltransferase activity in BM cells at concentrations as low as 1.5 mumol/L after a 1-hour exposure. O6-benzylguanine pretreatment markedly sensitized hematopoietic progenitor colony-forming cells to BCNU, resulting in a reduction in the dose of drug (termed the dose- modification factor) required to inhibit 50% of the colony formation (IC50) of threefold to fivefold. Since, unlike many other cell types, proliferating early (CD34+) hematopoietic precursors do not induce alkyltransferase, myelosuppression may be the dose-limiting toxicity of the combination of O6-benzylguanine plus BCNU in clinical trials.

scholarly journals Dissecting isoform selectivity of PI3K inhibitors: the role of non-conserved residues in the catalytic pocket

2008 ◽  
Vol 414 (3) ◽  
pp. 383-390 ◽  
Author(s):  
Mark Frazzetto ◽  
Cenk Suphioglu ◽  
Jiuxiang Zhu ◽  
Oleg Schmidt-Kittler ◽  
Ian G. Jennings ◽  
...  
Keyword(s):  
Point Mutations ◽  
Binding Pocket ◽  
Catalytic Site ◽  
Site Directed Mutagenesis ◽  
Specific Class ◽  
Pi3k Inhibitors ◽  
Isoform Selectivity ◽  
Phosphoinositide 3 Kinase ◽  
Conserved Amino Acids

The last few years have seen the identification of numerous small molecules that selectively inhibit specific class I isoforms of PI3K (phosphoinositide 3-kinase), yet little has been revealed about the molecular basis for the observed selectivities. Using site-directed mutagenesis, we have investigated one of the areas postulated as being critical to the observed selectivity. The residues Thr886 and Lys890 of the PI3Kγ isoform project towards the ATP-binding pocket at the entrance to the catalytic site, but are not conserved. We have made reciprocal mutations between those residues in the β isoform (Glu858 and Asp862) and those in the α isoform (His855 and Gln859) and evaluated the potency of a range of reported PI3K inhibitors. The results show that the potencies of β-selective inhibitors TGX221 and TGX286 are unaffected by this change. In contrast, close analogues of these compounds, particularly the α-isoform-selective compound (III), are markedly influenced by the point mutations. The collected data suggests two distinct binding poses for these inhibitor classes, one of which is associated with potent PI3Kβ activity and is not associated with the mutated residues, and a second that, in accord with earlier hypotheses, does involve this pair of non-conserved amino acids at the catalytic site entrance and contributes to the α-isoform-selectivity of the compounds studied.

scholarly journals Modulation of nitrosourea resistance in myeloid leukemias

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1487-1494 ◽  
Author(s):  
SL Gerson ◽  
JE Trey
Keyword(s):  
Dna Repair ◽  
Therapeutic Index ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Biochemical Modulation ◽  
Repair Protein ◽  
Myeloid Leukemias ◽  
Dna Repair Protein ◽  
Dna Alkyltransferase

Drug resistance in myeloid leukemias may be mediated by an increased capacity to repair chemotherapy-induced DNA damage. Some tumor cell lines that are resistant to nitrosoureas contain the DNA repair protein O6-alkylguanine-DNA alkyltransferase (alkyltransferase). This protects cells by removing cytotoxic, nitrosourea-induced O6-alkylguanine adducts. We measured the level of alkyltransferase activity in myeloid leukemic cells freshly obtained from patients to determine whether the alkyltransferase was an important factor in nitrosourea resistance in these cells and whether inactivation of this protein could sensitize leukemic cells to nitrosoureas. Myeloid leukemic cells from patients with acute nonlymphocytic leukemia and chronic myelogenous leukemia had higher levels of alkyltransferase than did myeloid precursors from normal donors (P less than .01). This difference did not appear to be due to the state of differentiation of the leukemic or normal cells. To show that this repair protein mediated nitrosourea resistance in leukemic cells, cells were treated with the modified base O6- methylguanine to selectively and irreversibly inactivate the alkyltransferase and then exposed to 1,3-bis (2-chloroethyl)-1- nitrosourea (BCNU). An 18-hour incubation in 0.5 mmol/L O6- methylguanine caused an 87% +/- 3.6% decrease in alkyltransferase activity in leukemic cells and a 73% +/- 8.6% decrease in normal myeloid precursors. After treatment with O6-methylguanine, clonogenic leukemic cells from ten different donors became much more sensitive to BCNU, with a decrease in the dose needed to reduce colony survival by 50% (LD50) of 6.3 +/- 1.4-fold. A lesser effect was seen on CFU-GM, BFU- E, and CFU-GEM where the LD50 decreased two- to threefold. These studies show that nitrosourea resistance in myeloid leukemic cells can be abrogated by inactivation of the DNA repair protein O6-alkylguanine- DNA alkyltransferase. This method of biochemical modulation of DNA repair will sensitize leukemic cells to nitrosoureas in vitro and has the potential of increasing the therapeutic index of nitrosoureas in this disease.

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