RESUMO
Human checkpoint kinase ataxia telangiectasia-mutated (ATM) plays a key role in initiation of the DNA damage response following DNA double-strand breaks. ATM inhibition is a promising approach in cancer therapy, but, so far, detailed insights into the binding modes of known ATM inhibitors have been hampered due to the lack of high-resolution ATM structures. Using cryo-EM, we have determined the structure of human ATM to an overall resolution sufficient to build a near-complete atomic model and identify two hitherto unknown zinc-binding motifs. We determined the structure of the kinase domain bound to ATPγS and to the ATM inhibitors KU-55933 and M4076 at 2.8 Å, 2.8 Å and 3.0 Å resolution, respectively. The mode of action and selectivity of the ATM inhibitors can be explained by structural comparison and provide a framework for structure-based drug design.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sítios de Ligação , Domínio Catalítico , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Morfolinas/química , Morfolinas/metabolismo , Mutação , Neoplasias/genética , Conformação Proteica , Pironas/química , Pironas/metabolismoRESUMO
Eubacterial tRNA-guanine transglycosylase (TGT) is involved in the hyper-modification of cognate tRNAs leading to the exchange of G34 at the wobble position in the anticodon loop by preQ1 (2-amino-5-(aminomethyl)pyrrolo[2,3-d]pyrimidin-4(3H)-one) as part of the biosynthesis of queuine (Q). Mutation of the tgt gene in Shigella flexneri results in a significant loss of pathogenicity of the bacterium, revealing TGT as a new target for the design of potent drugs against Shigellosis. The X-ray structure of Zymomonas mobilis TGT in complex with preQ1 was used to search for new putative inhibitors with the computer program LUDI. An initial screen of the Available Chemical Directory, a database compiled from commercially available compounds, suggested several hits. Of these, 4-aminophthalhydrazide (APH) showed an inhibition constant in the low micromolar range. The 1.95 A crystal structure of APH in complex with Z. mobilis TGT served as a starting point for further modification of this initial lead.
Assuntos
Desenho de Fármacos , Disenteria Bacilar/tratamento farmacológico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Guanina/análogos & derivados , Pentosiltransferases/antagonistas & inibidores , Shigella flexneri/enzimologia , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Bases de Dados como Assunto , Disenteria Bacilar/microbiologia , Inibidores Enzimáticos/metabolismo , Guanina/química , Guanina/metabolismo , Cinética , Modelos Moleculares , Estrutura Molecular , Pentosiltransferases/metabolismo , Ftalazinas/química , Ftalazinas/metabolismo , Ftalazinas/farmacologia , Pirimidinonas/química , Pirimidinonas/metabolismo , Pirróis/química , Pirróis/metabolismo , Shigella flexneri/efeitos dos fármacos , Software , Eletricidade Estática , Termodinâmica , Zymomonas/enzimologiaRESUMO
The tRNA modifying enzyme tRNA-guanine transglycosylase (TGT) is involved in the exchange of guanine in the first position of the anticodon with preQ1 as part of the biosynthesis of the hypermodified base queuine (Q). Mutation of Ser90 to an alanine in Escherichia coli TGT leads to a dramatic reduction of enzymatic activity (Reuter, K. et al. (1994) Biochemistry 33, 7041-7046). To further clarify the role of this residue in the catalytic center, we have mutated the corresponding Ser103 of the crystallizable Zymomonas mobilis TGT into alanine. The crystal structure of a TGT(S103A)/preQ1 complex combined with biochemical data presented in this paper suggest that Ser103 is essential for substrate orientation in the TGT reaction.