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

描述（由申请人提供）：在明确确定金属博来霉素（MBLM）与DNA的结合模式特征方面存在根本性差距。这一差距代表了一个重要的问题，因为药物的C-末端取代基（尾部），参与DNA结合的BLM，也被确定为肺毒性的关键因素归因于BLM。因此，这一差距阻碍了对这些抗癌药物的结构/毒性相关性的理解。长期目标是更好地了解生物相关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BLM产生不同水平肺纤维化的影响。本特定应用的目的是确定BLM尾部的化学结构如何影响FeIIBLM和DNA之间的结构相互作用，与DNA碱基序列无关，并考虑具有不同程度肺毒性的尾部。中心假设是具有不同化学结构的BLM尾将以特定的方式与DNA中的原子相关联，并产生相应MBLM-DNA复合物的（C-末端）- DNA区域的不同溶液结构。我们已经制定了这一假设的基础上，初步的研究结果表明，BLM尾可以锚MBLM的DNA，并影响的方式，其中其余的C-末端的药物是定位在DNA螺旋。拟议研究的基本原理是，评估拟议导致小鼠不同水平肺纤维化的DNA结合模式BLM将达到两个目的：1）更好地建立MBLM和DNA之间分子识别的结构基础，以及2）帮助描绘这些药物的结构/毒性相关性。在强有力的初步数据的指导下，这一假设将通过追求两个特定的目标来检验：1）使用两个不同的DNA片段和具有不同结构和毒性的尾部的相关MBLM来鉴定溶液中MBLM和DNA之间的相互作用，每个DNA片段包含特定的BLM结合位点; 2）通过对MBLM-DNA复合物的分子模拟，表征BLM尾在MBLM与DNA之间的结构相互作用中的作用。根据第一个目标，NMR光谱应用于顺磁性分子，如生物相关的MBLM（FeIIBLM），将被用来确定考虑和DNA的MBLM之间的原子-原子接触。在第二个目标下，分子动力学计算将被用来找到所考虑的MBLM-DNA复合物的溶液结构。这种方法在申请人手中已被确定为可行的。拟议的研究具有创新性，因为它只考虑了生物相关的MBLM，由于其顺磁性质，从结构角度来看，MBLM尚未受到足够的关注，并且它分离了影响BLM与DNA结合的因素。这一贡献将是重要的，因为它有望从结构的角度垂直推进和扩大对毒性的理解，这是指导开发对癌症患者具有较轻副作用的BLM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.