Theoretical study of ribonucleotide reductase mechanism-based inhibition by 2'-azido-2'-deoxyribonucleoside 5'-diphosphates

2'-azido-2'-deoxyribonucleoside 5'-diphosphates are mechanism-based inhibitors of Ribonucleotide Reductase. Considerable effort has been made to elucidate their mechanism of inhibition, which is still controversial and not fully understood. Previous studies have detected the formation of a radical intermediate when the inhibitors interact with the enzyme, and several authors have proposed possible structures for this radical. We have conducted a theoretical study of the possible reactions involved, which allowed us to identify the structure of the new radical among the several proposals. A new reactional path is also proposed that is the most kinetically favored to yield this radical and ultimately inactivate the enzyme. The energetic involved in this mechanism, both for radical formation and radical decay, as well as the calculated Hyperfine Coupling Constants for the radical intermediate, are in agreement with the correspondent experimental values. This mechanistic alternative is fully coherent with remaining experimental data.     

Mechanism for ribonucleotide reductase inactivation by the anticancer drug gemcitabine

Gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) is a very promising anticancer drug, already approved for clinical use in three therapeutic indications. It is metabolized intracellularly to 5'-diphosphate (dFdCDP), which is known to be a potent inhibitor of ribonucleotide reductase (RNR). Although several nucleotide analogs show in vitro capacity of RNR inactivation, none has shown the in vivo efficacy of dFdCDP. Accordingly, the experimental data suggests that its mechanism of inhibition is different from the other known RNR suicide inhibitors. Enzyme inhibition in the absence of reductive species leads to complete loss of the essential radical in subunit R2, and formation of a new nucleotide-based radical. Interestingly, however, the presence of the reductants does not prevent inhibition--the radical is not lost but the targeted subunit of RNR becomes R1, which is inactivated possibly by alkylation. We have conducted a theoretical study, which led us to the first proposal of a possible mechanism for RNR inhibition by dFdCDP in the absence of reductants. This mechanism turned out to be very similar to the natural substrate reduction pathway and only deviates from the natural course after the formation of the well-known disulphide bridge. This deviation is caused precisely by the F atom in the beta-face, only present in this inhibitor. The essential radical in R2 is lost, and so is the enzyme catalytic activity. The nucleotide-based radical that constitutes the end product of our mechanism has been suggested in the literature as a possible candidate for the one detected experimentally. In fact, all experimental data available has been reproduced by the theoretical calculations performed here.     

利用噬菌体展示技术制备识别酵母RNR_3原核表达片段的单链抗体

选取酵母细胞RNR_3蛋白序列中高保守片段:145-298位氨基酸序列,命名为RNR_3h进行原核表达.并利用噬菌体展示技术从非免疫小鼠噬菌体展示抗体库中淘选到能与该原核表达产物特异性结合的单链抗体.得到的3种单链抗体均识别原核表达片段的空间抗原位点.为寻找酵母RNR1和RNR_3的诱导表达水平与DNA损伤信号的相互关系,将真核生物RNRs发展为检测环境化学致痛物DNA损伤效应的生物标志物进行了尝试.