Characterisation of the molecular structure and function of topoisomerase I using anticancer agents as molecular probes acting in a new allosteric binding-site

前言：拓扑异构酶（Topo）是治疗多种癌症的重要靶点。为此，这些酶的抑制剂是最广泛使用的抗癌药物之一。例如，Topo I是维持我们遗传密码完整性所必需的酶，以避免引入有害的突变，而这些酶的抑制剂诱导该密码的破坏和细胞的死亡。作为一名独立的研究人员，我已经开发了近两年的一个新的有前途的抗癌药物家族，称为“N-苯基脲基苯磺酸盐”（PUB-SO），阻断这种酶的活性。PUB-SO与临床或开发中使用的已知Topo I抑制剂不同。它们基于一种真正的新分子模板，易于制备和修饰，以低成本生产有效的抗癌药物。事实上，我已经证明，这些化合物通过一种机制阻断拓扑异构酶I的活性，这种机制尚未确定，但与临床使用的药物不同。这表明，它们可能不会受到腹泻和中性粒细胞减少症等相同副作用的困扰，以及与其化学不稳定性和诱导耐药性有关的其他问题。研究计划：我的研究计划旨在定位和研究PUB-SO与Topo I结合的位点。为此，我将使用PUB-SO衍生物和类似物来研究3D中的结合位点。目的1：设计和制备PUB-SOs衍生物和类似物，用作“特殊探针”。目的2：定位Topo I结合PUB-SOs的位点，阻断其对遗传密码的修复活性。目标3：使用生物学和药理学工具研究这种结合位点，例如测量新化合物对Topo I修复活性和癌细胞繁殖的影响。目的4：构建Topo I的3D计算机模型，其中PUB-SO被固定，以了解这些分子的作用机制。目的5：设计和开发一种新的测试，以快速鉴定抑制Topo I活性的有前途的化合物。结论：我的研究计划是一个独特的机会，在短期内扩大我们对拓扑I的三维结构的理解，当它与PUB-SO形成复合物时。这些研究为快速鉴定抑制Topo I的新分子提供了新的方法。最后，这项研究计划将允许在更长的时间内开发新的工具，用于设计和开发下一代抑制剂和抗癌药物。我的研究计划还将为药物设计部门的高素质人员提供激励性的多学科培训，特别是在有机合成，分子药理学，细胞生物学，质谱和分子建模方面。

INTRODUCTION: Topoisomerases (Topo) are important therapeutic targets to treat many cancers. To that end, inhibitors of these enzymes are amongst the most widely used anticancer drugs. For example, Topo I is an enzyme essential to maintain the integrity of our genetic code to avoid introducing harmful mutations while inhibitors of these enzymes induce breakages of this code and death of the cells. I have developed for nearly two years as an independent researcher a new family of promising anticancer drugs referred to “N-phenyl ureidobenzenesulfonates” (PUB-SOs) blocking the activity of this enzyme. PUB-SOs are different from known Topo I inhibitors used in clinic or in development. They are based on a genuine new molecular template easily prepared and modifiable to produce efficient anticancer drugs at low costs. Indeed, I have shown that these compounds block the activity of Topo I by a mechanism that still not identified but different from the drugs used in the clinic. This suggests that they might not be plagued with the same side effects such as diarrhoea and neutropenia and other problems related to their chemical instability and induction of chemoresistance. RESEARCH PROGRAM: My research program aims to locate and study the site where PUB-SOs binds to Topo I. To that end, I will use PUB-SO derivatives and analogs to study that binding site in 3D. Aim 1: Design and preparation of PUB-SOs derivatives and analogs that will be used as “special probes”. Aim 2: Localization of the site on Topo I binding PUB-SOs to block its repairing activity of the genetic code. Aim 3: Study this binding site using tools of biology and pharmacology such as a measure of the effect of new compounds on the repair activity of Topo I and the reproduction of cancer cells. Aim 4: Construction of 3D computer models of Topo I where the PUB-SOs are fixed in order to understand the mechanism of action of these molecules. Aim 5: Design and development of a new test to identify quickly promising compounds inhibiting the activity of Topo I. CONCLUSION: My research program is a unique opportunity to expand on a short-term level our understanding of the tridimensional structure of Topo I when it forms complexes with PUB-SOs. These studies provide a new method for rapid identification of new molecules of interest inhibiting Topo I. Finally, this research program will allow on a longer term to develop new tools for the design and development of the next generations of inhibitors and anticancer drugs. My research program will also offer a stimulating multidisciplinary training for highly qualified personnel in the drug design sector, notably in organic synthesis, molecular pharmacology, cellular biology, mass spectrometry and molecular modeling.