Asthana Shailendra, Shukla Saumya, Ruggerone Paolo, Vargiu Attilio V
Dipartimento di Fisica, Università degli Studi di Cagliari, Cittadella Universitaria , 09042 Monserrato, CA, Italy.
Biochemistry. 2014 Nov 11;53(44):6941-53. doi: 10.1021/bi500490z. Epub 2014 Oct 23.
Recently, we reported on a potent benzimidazole derivative (227G) that inhibits the growth of the bovine viral diarrhea virus (BVDV) in cell-based and enzyme assays at nanomolar concentrations. The target of 227G is the viral RNA-dependent RNA polymerase (RdRp), and the I261M mutation located in motif I of the RdRp finger domain was found to induce drug resistance. Here we propose a molecular mechanism for the retained functionality of the enzyme in the presence of the inhibitor, on the basis of a thorough computational study of the apo and holo forms of the BVDV RdRp either in the wild type (wt) or in the form carrying the I261M mutation. Our study shows that although the mutation affects to some extent the structure of the apoenzyme, the functional dynamics of the protein appear to be largely maintained, which is consistent with the retained functionality of this natural mutant. Despite the binding site of 227G not collapsing or undergoing drastic structural changes upon introduction of the I261M substitution, these alterations reflect crucially on the binding mode of 227G, which is significantly different from that found in wt RdRp. In particular, while in the wt system the four loops lining the template entrance site embrace 227G and close the template passageway, in the I261M variant the template entrance is only marginally occluded, allowing in principle the translocation of the template to the interior of the enzyme. In addition, the mutated enzyme in the presence of 227G retains several characteristics of the wt apoprotein. Our work provides an original molecular picture of a resistance mechanism that is consistent with published experimental data.
最近，我们报道了一种强效苯并咪唑衍生物（227G），它在基于细胞和酶的检测中，能以纳摩尔浓度抑制牛病毒性腹泻病毒（BVDV）的生长。227G的作用靶点是病毒RNA依赖性RNA聚合酶（RdRp），并且发现位于RdRp指状结构域基序I中的I261M突变会诱导耐药性。在此，我们基于对野生型（wt）或携带I261M突变形式的BVDV RdRp的无配体和全酶形式进行的深入计算研究，提出了在存在抑制剂的情况下该酶保留功能的分子机制。我们的研究表明，尽管该突变在一定程度上影响了无配体酶的结构，但蛋白质的功能动力学似乎在很大程度上得以维持，这与该天然突变体保留的功能是一致的。尽管引入I261M替代后227G的结合位点没有塌陷或发生剧烈的结构变化，但这些改变对227G的结合模式有至关重要的影响，这与在wt RdRp中发现的结合模式显著不同。特别是，在wt系统中，位于模板入口位点的四个环围绕着227G并封闭了模板通道，而在I261M变体中，模板入口只是略微被阻塞，原则上允许模板向酶内部转运。此外，存在227G时的突变酶保留了wt无配体蛋白的几个特征。我们的工作提供了一种与已发表的实验数据一致的耐药机制的原始分子图景。
