Role of the bleomycin C-termini in DNA binding and toxicity by metallo-bleomycins

描述(申请人提供)：在明确建立金属博莱菌素(MBLms)与DNA的结合模式特征方面存在着根本的差距。这一差距代表了一个重要的问题，因为参与BLM与DNA结合的药物的C末端取代基(Tail)也被认为是BLMS肺毒性的关键因素。因此，这一差距阻碍了对这些抗癌药物结构/毒性相关性的了解。长期目标是更好地了解生物相关的MBLM与DNA的结合，通过一种可以分离影响它的各种因素的方法，并专注于这种结合中的尾部对产生不同程度的肺纤维化的BLM的影响。这一特殊应用的目的是确定BLM尾巴的化学结构如何影响FeIIBLM与DNA之间的结构相互作用，独立于DNA碱基序列，并考虑不同程度的肺毒性。中心假设是，具有不同化学结构的BLM尾巴会以特定的方式与DNA中的原子相互作用，并为相应的MBLM-DNA复合物产生不同的(C-末端)-DNA区域的溶液结构。我们基于初步发现提出了这一假设，这些发现表明BLM尾部可以将MBLM锚定在DNA上，并影响药物的其余C末端在DNA螺旋中的定位方式。这项研究的基本原理是，评估导致小鼠不同程度肺纤维化的DNA结合模式BLMS将有两个目的：1)更好地建立MBLM和DNA之间的分子识别的结构基础；2)帮助描述这些药物的结构/毒性相关性。在强大的初步数据的指导下，这一假说将通过追求两个具体目标来验证：1)使用两个不同的DNA片段(每个片段包含一个特定的BLM结合部位)以及相关的MBLM(其尾部具有不同的结构和毒性)来确定MBLM与DNA之间的相互作用；以及2)通过对所考虑的MBLM-DNA复合体的分子建模来表征BLM尾部在MBLM与DNA之间结构相互作用中的作用。在第一个目标下，核磁共振波谱应用于顺磁性分子，如生物相关的MBLMS(FeIIBLMS)，将被用于鉴定所考虑的MBLMS与DNA之间的原子-原子接触。在第二个目标下，将使用分子动力学计算来寻找所考虑的MBLM-DNA络合物的溶液结构。这一做法已在申请人手中确立为可行。这项研究具有创新性，因为它只考虑了与生物相关的MBLM，由于它们的顺磁性，从结构角度看没有得到足够的关注，并且它分离了影响BLMS与DNA结合的因素。这一贡献将是重大的，因为它有望从结构角度垂直推进和扩大对毒性的理解，这是指导癌症患者开发副作用较轻的博莱曼类似物所必需的。

DESCRIPTION (provided by applicant): There is a fundamental gap in definitely establishing the binding mode characteristic of metallo-bleomycins (MBLMs) to DNA. This gap represents an important problem, since the drugs' C-terminus substituents (tails), which participate on DNA binding by BLM, have also been identified as key factors in the pulmonary toxicity attributed to BLMs. Therefore, this gap hinders the understanding of structure/toxicity correlations for these anticancer drugs. The long-term goal is to better understand the binding of biologically relevant MBLMs to DNA, through an approach that can separate the various factors that affect it, and focusing on the impact of the tails in this binding for BLMs producing different levels of pulmonary fibrosis. The objective in this particular application is to determine how the chemical structures of the BLM tails influence structural interactions between FeIIBLM and DNA, independently of the DNA base sequence, and considering tails with different degrees of pulmonary toxicity. The central hypothesis is that BLM tails with different chemical structures wil correlate with the atoms in DNA in specific ways, and generate different solution structures of the (C-terminus)- DNA regions for the corresponding MBLM-DNA complexes. We have formulated this hypothesis based upon preliminary findings that suggest that the BLM tail can anchor MBLM to DNA, and affect the way in which the rest of the C-terminus of the drug is positioned in the DNA helix. The rationale for the proposed research is that assessing the DNA-binding modes BLMs proposed to cause various levels of pulmonary fibrosis in mice will serve two purposes: 1) to better establish the structural basis for molecular recognition between MBLM and DNA, and 2) to help delineate structure/toxicity correlations for these drugs. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Identify interactions between MBLM and DNA in solution using two distinct DNA segments, each containing a specific BLM binding site, and relevant MBLMs with tails with different structures and toxicities; and 2) Characterize the effect of the BLM tail in the structural interactions between MBLM and DNA through molecular modeling of the MBLM-DNA complexes considered. Under the first aim, NMR spectroscopy applied to paramagnetic molecules such as the biologically relevant MBLMs (FeIIBLMs), will be used to identify atom-atom contacts between the MBLMs considered and DNA. Under the second aim, molecular dynamics calculations will be used to find the solution structures of the MBLM-DNA complexes considered. This approach has been established as feasible in the applicant's hands. The proposed research is innovative because it only considers biologically relevant MBLMs, which have no received sufficient attention from the structural point of view due to their paramagnetic nature, and it isolates the factors affecting DNA binding by BLMs. This contribution will be significant because it is expected to vertically advance and expand the understanding of toxicity from the structural point of view, required to guide the development of BLM analogs with milder side effects for cancer patients.