Abstract:The role of lysine(165) in the activity of the DNA repair protein, O(6)-alkylguanine-DNA alkyltransferase (AGT), and the ability of AGT to react with the pseudosubstrate inhibitor, O(6)-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 … Show more
“…At the third step, transfer of the methyl group of O6mG to the sulfur atom of the Cys145 thiolate anion would occur simultaneously with migration of Lys165 proton to Tyr114 (Figure ). Hence the presence of Tyr114 and Lys165 in the catalytic cycle are important for the demethylation of O6mG, which is in agreement with mutational studies. , Thus, the present study provides new interesting information related to repair of alkylated DNA by AGT, which may be useful to design potent AGT inhibitors.…”
O6-alkylguanine-DNA alkyltransferase (AGT) repairs O6-methylguanine (O6mG) in DNA that is known to cause mutation and cancer. On the basis of calculations performed using density functional theory involving the active site of AGT, a mechanism for catalytic demethylation of O6mG to guanine has been proposed. In this mechanism, roles of six amino acids, i.e., Cys145, His146, Glu172, Tyr114, Lys165, and Ser159 in catalytic demethylation of O6mG are involved. This mechanism has three steps as follows. At the first step, Cys145 in the Cys145-water-His146-Glu172 tetrad is converted to cysteine thiolate anion while at the second step, abstraction of the Tyr114 proton by the N3 site of O6mG occurs in a barrierless manner. In the third step, abstraction of Lys165 proton by deprotonated Tyr114 and transfer of the methyl group of O6mG to the thiolate group of Cys145 anion occur simultaneously. As AGT is a major target in cancer therapy, identification of the roles of the different amino acids in demethylation of O6mG is expected to be useful in designing efficient AGT inhibitors.
“…At the third step, transfer of the methyl group of O6mG to the sulfur atom of the Cys145 thiolate anion would occur simultaneously with migration of Lys165 proton to Tyr114 (Figure ). Hence the presence of Tyr114 and Lys165 in the catalytic cycle are important for the demethylation of O6mG, which is in agreement with mutational studies. , Thus, the present study provides new interesting information related to repair of alkylated DNA by AGT, which may be useful to design potent AGT inhibitors.…”
O6-alkylguanine-DNA alkyltransferase (AGT) repairs O6-methylguanine (O6mG) in DNA that is known to cause mutation and cancer. On the basis of calculations performed using density functional theory involving the active site of AGT, a mechanism for catalytic demethylation of O6mG to guanine has been proposed. In this mechanism, roles of six amino acids, i.e., Cys145, His146, Glu172, Tyr114, Lys165, and Ser159 in catalytic demethylation of O6mG are involved. This mechanism has three steps as follows. At the first step, Cys145 in the Cys145-water-His146-Glu172 tetrad is converted to cysteine thiolate anion while at the second step, abstraction of the Tyr114 proton by the N3 site of O6mG occurs in a barrierless manner. In the third step, abstraction of Lys165 proton by deprotonated Tyr114 and transfer of the methyl group of O6mG to the thiolate group of Cys145 anion occur simultaneously. As AGT is a major target in cancer therapy, identification of the roles of the different amino acids in demethylation of O6mG is expected to be useful in designing efficient AGT inhibitors.
“…Tyr114, conserved in all known sequences, was shown to play a key role in substrate specificity, DNA binding, and protection against mutations induced by methylating agents . Cells expressing the K165A mutant of MGMT were still protected by the enzyme from the mutagenic effects of O 6 -methylating agents and could not be sensitized by O 6 -BG . Similar results were obtained for various Gly156 mutants as well as for mutations at Cys150, Asn157, Tyr158, Ser159, Leu168, and Leu169. , At codon 160 mutations can lead to either increased sensitivity (G160A, G160W) or resistance toward O 6 -BG (G160R and many others) .…”
Section: Introductionsupporting
confidence: 58%
“…The strongest interactions involved Tyr158, Arg135, and Asn137 (−1.5, −1.2, −1.1 kcal/mol), consistent with the geometry of S -benzyl MGMT discussed above. Weaker interactions of the O 6 substituent of 20 were observed with Pro140, Ser159, and Gly160 (−1.1, −1.0, −0.7 kcal/mol), whose importance in the binding of O 6 -BG ( 8 ) has been discussed in several publications. ,,, The weaker interactions of 20 can be rationalized on the basis that in the complex the mean distances between the 4-BT group (center of ring) and the C α of Pro140, Ser159, and Gly160 range from 5.7 to 5.9 Å and are 0.5−1.4 Å larger than the corresponding distances in S -benzyl MGMT. 2 Definition of the structural subunits of MGMT inhibitors (example: compound 24 ) for which enzyme:inhibitor interaction analysis was performed by molecular modeling.…”
Section: Resultsmentioning
confidence: 96%
“…Site-directed mutagenesis studies indicated that several amino acid residues are involved in the MGMT reaction mechanism. The residues Arg128, Tyr114, and Lys165 were found to be necessary for DNA binding, − while Pro140, Gly156, Tyr158, and Gly160 proved to be essential for reaction with either O 6 -BG or oligonucleotides containing O 6 -methylguanine. Tyr114, conserved in all known sequences, was shown to play a key role in substrate specificity, DNA binding, and protection against mutations induced by methylating agents .…”
A series of potential inhibitors of the human DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) were synthesized, characterized in detail by NMR, and tested for their ability to deplete MGMT activity in vitro. The new compounds, omega-[O(6)-R-guan-9-yl]-(CH(2))(n)-beta-d-glucosides with R = benzyl or 4-bromothenyl and omega = n = 2, 4,. 12, were compared with the established inhibitors O(6)-benzylguanine (O(6)-BG), 8-aza-O(6)-benzylguanine (8-aza-BG), and O(6)-(4-bromothenyl)guanine (4-BTG), which exhibit in an in vitro assay IC(50) values of 0.62, 0.038, and 0.009 microM, respectively. Potential advantages of the glucosides are improved water solubility and selective uptake in tumor cells. The 4-BTG glucosides with n = 2, 4, 6 show moderate inhibition with an IC(50) of ca. 0.5 microM, while glucosides derived from BG and 8-aza-BG showed significantly poorer inhibition compared to the parent compounds. The 4-BTG glucosides with n = 8, 10, 12 were effective inhibitors with IC(50) values of ca. 0.03 microM. To understand this behavior, extensive molecular modeling studies were performed using the published crystal structure of MGMT (PDB entry: ). The inhibitor molecules were docked into the BG binding pocket, and molecular dynamics simulations with explicit water molecules were carried out. Stabilization energies for the interactions of specific regions of the inhibitor and individual amino acid residues were calculated. The alkyl spacer is located in a cleft along helix 6 of MGMT. With increasing spacer length there is increasing interaction with several amino acid residues which play an important role in the proposed nucleotide flipping mechanism required for DNA repair.
“…The allowed torsion angles of non‐glycine residues require backbone conformational changes that prabably distort the adjacent loop (residues 158–160), which defines one wall of the benzyl‐binding pocket. Similarly, all 19 mutations generated at Lys165 significantly increase resistance to O 6 ‐BG (Xu‐Welliver et al ., 2000) since the lysine side chain participates in a hydrogen bond with the carbonyls of Val148, Val155 and Asn157, stabilizing this active site loop (Figure 3B).…”
Human O(6)-alkylguanine-DNA alkyltransferase (AGT), which directly reverses endogenous alkylation at the O(6)-position of guanine, confers resistance to alkylation chemotherapies and is therefore an active anticancer drug target. Crystal structures of active human AGT and its biologically and therapeutically relevant methylated and benzylated product complexes reveal an unexpected zinc-stabilized helical bridge joining a two-domain alpha/beta structure. An asparagine hinge couples the active site motif to a helix-turn-helix (HTH) motif implicated in DNA binding. The reactive cysteine environment, its position within a groove adjacent to the alkyl-binding cavity and mutational analyses characterize DNA-damage recognition and inhibitor specificity, support a structure-based dealkylation mechanism and suggest a molecular basis for destabilization of the alkylated protein. These results support damaged nucleotide flipping facilitated by an arginine finger within the HTH motif to stabilize the extrahelical O(6)-alkylguanine without the protein conformational change originally proposed from the empty Ada structure. Cysteine alkylation sterically shifts the HTH recognition helix to evidently mechanistically couple release of repaired DNA to an opening of the protein fold to promote the biological turnover of the alkylated protein.
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