RESUMO
Ribonucleic acid (RNA) is central to many life processes and, to fulfill its function, it has a substantial chemical variety in its building blocks. Enzymatic thiolation of uridine introduces 4-thiouridine (s4 U) into many bacterial transfer RNAs (tRNAs), which is used as a sensor for UV radiation. A similar modified nucleoside, 2-thiocytidine, was recently found to be sulfur-methylated especially in bacteria exposed to antibiotics and simple methylating reagents. Herein, we report the synthesis of 4-methylthiouridine (ms4 U) and confirm its presence and additional formation under stress in Escherichia coli. We used the synthetic ms4 U for isotope dilution mass spectrometry and compared its abundance to other reported tRNA damage products. In addition, we applied sophisticated stable-isotope pulse chase studies (NAIL-MS) and showed its AlkB-independent removal inâ vivo. Our findings reveal the complex nature of bacterial RNA damage repair.
Assuntos
Escherichia coli/metabolismo , RNA Bacteriano/metabolismo , RNA de Transferência/metabolismo , Tiouridina/metabolismo , Modelos Moleculares , Estrutura Molecular , RNA Bacteriano/química , RNA de Transferência/química , Tiouridina/síntese química , Tiouridina/químicaRESUMO
A model system of two related enzymes with conserved binding sites, namely N-myristoyltransferase from two different organisms, was studied to decipher the driving forces that lead to selective inhibition in such cases. Using a combination of computational and experimental tools, two different selectivity-determining features were identified. For some ligands, a change in side-chain flexibility appears to be responsible for selective inhibition. Remarkably, this was observed for residues orienting their side chains away from the ligands. For other ligands, selectivity is caused by interfering with a water molecule that binds more strongly to the off-target than to the target. On the basis of this finding, a virtual screen for selective compounds was conducted, resulting in three hit compounds with the desired selectivity profile. This study delivers a guideline on how to assess selectivity-determining features in proteins with conserved binding sites and to translate this knowledge into the design of selective inhibitors.
Assuntos
Aciltransferases/antagonistas & inibidores , Aciltransferases/metabolismo , Desenho de Fármacos , Leishmania major/metabolismo , Modelos Moleculares , Aciltransferases/química , Sítios de Ligação/fisiologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Leishmania major/enzimologia , LigantesRESUMO
Golgi α-mannosidase II (GMII) is a glycoside hydrolase playing a crucial role in the N-glycosylation pathway. In various tumour cell lines, the distribution of N-linked sugars on the cell surface is modified and correlates with the progression of tumour metastasis. GMII therefore is a possible molecular target for anticancer agents. Here, we describe the identification of a non-competitive GMII inhibitor using computer-aided drug design methods including identification of a possible allosteric binding site, pharmacophore search and virtual screening.