DNA death grip Poly(ADP-ribose) polymerase–1 (PARP-1) binds to DNA breaks and recruits DNA repair components. Cancer-killing PARP-1 inhibitor (PARPi) compounds all block the same catalytic site but exhibit vastly different efficacy. Zandarashvili et al. investigated the molecular impact of PARPi binding to PARP-1 (see the Perspective by Slade and Eustermann). Different PARPi molecules perturb PARP-1 allostery in diverse manners: Some drive allostery to promote release of PARP-1 from DNA, and others drive allostery to promote retention. These insights help explain the different efficacies in the clinic and enable conversion of a pro-release, ineffective cancer-killing compound to a pro-retention, more effective PARPi. Science, this issue p. eaax6367; see also p. 30 Different poly(ADP-ribose) polymerase–1 (PARP-1) inhibitors used to treat cancer either trap or release PARP-1 at DNA break sites. INTRODUCTION Poly(ADP-ribose) polymerase–1 (PARP-1) is an abundant enzyme in the cell nucleus that regulates genome repair by binding to DNA damage sites and creating the poly(ADP-ribose) posttranslational modification. PARP-1 hyperactivity leads to cell stress or death associated with many prominent diseases (e.g., cardiovascular disease and several common neurodegenerative disorders). PARP-1 has notably emerged as an effective clinical target for a growing list of cancers. Clinical PARP-1 inhibitor (PARPi) compounds all bind at the same location at the catalytic center of the enzyme to block the binding of substrate nicotinamide adenine dinucleotide (NAD+) and prevent poly(ADP-ribose) production, yet they exhibit vastly different outcomes in tumor cell killing and efficacy in the clinic—a paradox that has confounded the development of PARPi. The resolution of this paradox likely lies in the realization that the most effective PARPi compounds trap PARP-1 at the site of a DNA break, generating a lesion that becomes cytotoxic, especially in tumor cells with deficiencies in the repair of DNA strand breaks. RATIONALE The molecular roots of PARP-1 trapping on DNA remain poorly understood. We focused on the retention of PARP-1 on damaged DNA, examining a panel of PARPi that included those currently approved for clinical use. Solution biophysical approaches, especially hydrogen/deuterium exchange mass spectrometry (HXMS), combined with x-ray structures and a battery of biochemical assays, were used to interrogate the molecular impact of PARPi binding to PARP-1 engaged on sites of DNA damage. Structure-guided modification of PARPi through medicinal chemistry was combined with chromatin fractionation to monitor trapped PARP-1 and with cell survival assays to assess PARPi efficacy, so as to probe the molecular underpinnings of the variable outcomes between clinical PARPi. RESULTS HXMS experiments revealed that a critical allosteric regulatory domain of PARP-1, the helical domain (HD), is affected in distinct ways depending on the particular PARPi engaged in the NAD+-binding site adjacent to the HD. Certain PARPi destabilized specific HD regions, some had no effect on the HD, and others actually stabilized regions of the HD. PARPi that destabilized the HD increased PARP-1 affinity for DNA and retained PARP-1 on DNA breaks. Conversely, PARPi that stabilized the HD decreased PARP-1 affinity for DNA breaks. PARPi molecules were thus classified into three types: type I, allosteric pro-retention on DNA; type II, non-allosteric; and type III, allosteric pro-release from DNA. X-ray structure analysis identified PARPi contacts with the HD structural element helix αF, which was established to be the discriminating factor between the types of PARPi. We found that type I PARPi contact helix αF to initiate an allosteric chain reaction that travels ~40 Å through the multidomain PARP-1 molecule and culminates in increased DNA binding affinity. Structure-guided mutagenesis of helix αF disrupted PARPi contacts and abrogated the allosteric effects of a type I inhibitor, transforming it into a non-allosteric type II inhibitor. Other mutations that disrupted PARP-1 allostery, including one identified in a de novo PARPi-resistant patient with ovarian cancer, also prevented type I PARPi from retaining PARP-1 on a DNA break. Type III PARPi influenced PARP-1 allostery in a manner that reduced DNA binding and favored DNA release. Structure-inspired modification of a pro-release (type III) inhibitor converted it to a pro-retention (type I) inhibitor that conferred potent PARP-1 trapping within the cellular context and increased its ability to kill cancer cells. CONCLUSION Our findings establish the impact of clinical PARPi on PARP-1 allostery and demonstrate that allostery plays a critical role in cellular PARP-1 trapping and can increase potency toward cancer cell killing. The results illuminate the molecular basis for the fine-tuning of PARPi to achieve allosteric effects and to influence PARP-1 retention on DNA damage and trapping on chromatin in cells. In contrast to cancer, other diseases would seem to benefit from PARP-1 inhibition but not cell death (e.g., cardiovascular disease, neurodegenerative diseases, and inflammation). Our studies provide the molecular understanding and the appropriate toolset to create and evaluate tunable PARPi for clinical applications where PARP-1 trapping and associated cytotoxicity are either desirable or undesirable in specific patients. PARPi impact on PARP-1 allostery. PARP-1 (tan) uses multiple domains to detect DNA breaks, and DNA damage detection is allosterically coupled to poly(ADP-ribose) production. PARPi bind to the catalytic domain to inhibit PARP-1 activity. Type I PARPi influence PARP-1 allostery and retain PARP-1 on DNA (left, UKTT15 in green), whereas type III PARPi perturb PARP-1 allostery and release PARP-1 from DNA (right, veliparib in red). Type II PARPi do not influence PARP-1 allostery. The success of poly(ADP-ribose) polymerase–1 (PARP-1) inhibitors (PARPi) to treat cancer relates to their ability to trap PARP-1 at the site of a DNA break. Although different forms of PARPi all target the catalytic center of the enzyme, they have variable abilities to trap PARP-1. We found that several structurally distinct PARPi drive PARP-1 allostery to promote release from a DNA break. Other inhibitors drive allostery to retain PARP-1 on a DNA break. Further, we generated a new PARPi compound, converting an allosteric pro-release compound to a pro-retention compound and increasing its ability to kill cancer cells. These developments are pertinent to clinical applications where PARP-1 trapping is either desirable or undesirable.

中文翻译：

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DNA 断裂时变构 PARP-1 保留的结构基础

DNA 死亡之握聚（ADP-核糖）聚合酶-1 (PARP-1) 与 DNA 断裂结合并招募 DNA 修复成分。抗癌 PARP-1 抑制剂 (PARPi) 化合物都阻断相同的催化位点，但表现出截然不同的功效。赞达拉什维利等。研究了 PARPi 与 PARP-1 结合的分子影响（参见 Slade 和 Eustermann 的观点）。不同的 PARPi 分子以不同的方式干扰 PARP-1 变构：一些驱动变构促进 PARP-1 从 DNA 中释放，而另一些驱动变构促进保留。这些见解有助于解释临床中的不同功效，并能够将促释放、无效的杀癌化合物转化为促保留、更有效的 PARPi。科学，这个问题 p。eaax6367; 另见第 30 种不同的聚（ADP-核糖）聚合酶-1 (PARP-1) 抑制剂用于治疗癌症，可以在 DNA 断裂位点捕获或释放 PARP-1。简介 聚（ADP-核糖）聚合酶-1 (PARP-1) 是细胞核中一种丰富的酶，它通过与 DNA 损伤位点结合并产生聚（ADP-核糖）翻译后修饰来调节基因组修复。PARP-1 过度活跃会导致与许多主要疾病（例如，心血管疾病和几种常见的神经退行性疾病）相关的细胞应激或死亡。PARP-1 已成为越来越多癌症的有效临床靶点。临床 PARP-1 抑制剂 (PARPi) 化合物都结合在酶催化中心的同一位置，以阻断底物烟酰胺腺嘌呤二核苷酸 (NAD+) 的结合并阻止聚 (ADP-核糖) 的产生，然而，它们在肿瘤细胞杀伤和临床疗效方面表现出截然不同的结果——这一悖论混淆了 PARPi 的发展。这个悖论的解决可能在于认识到最有效的 PARPi 化合物会在 DNA 断裂位点捕获 PARP-1，从而产生具有细胞毒性的损伤，尤其是在 DNA 链断裂修复缺陷的肿瘤细胞中。基本原理 PARP-1 捕获在 DNA 上的分子根源仍然知之甚少。我们专注于 PARP-1 在受损 DNA 上的保留，检查了一组 PARPi，其中包括目前批准用于临床使用的那些。解决方案生物物理方法，尤其是氢/氘交换质谱 (HXMS)，结合 X 射线结构和一系列生化分析，用于研究 PARPi 与参与 DNA 损伤位点的 PARP-1 结合的分子影响。通过药物化学对 PARPi 进行结构引导修饰，结合染色质分离来监测捕获的 PARP-1，并结合细胞存活分析来评估 PARPi 功效，从而探索临床 PARPi 之间可变结果的分子基础。结果 HXMS 实验表明，PARP-1 的一个关键变构调节域，即螺旋域 (HD)，根据参与 HD 附近 NAD+ 结合位点的特定 PARPi 以不同方式受到影响。某些 PARPi 使特定的 HD 区域不稳定，有些对 HD 没有影响，而另一些实际上稳定了 HD 的区域。使 HD 不稳定的 PARPi 增加了 PARP-1 对 DNA 的亲和力，并在 DNA 断裂时保留了 PARP-1。反过来，稳定 HD 的 PARPi 降低了 PARP-1 对 DNA 断裂的亲和力。PARPi 分子因此分为三种类型：I 型，DNA 上的变构促保留；II型，非变构；和 III 型，从 DNA 中变构促释放。X 射线结构分析确定了 PARPi 与 HD 结构元素螺旋 αF 的接触，这被确定为 PARPi 类型之间的区分因素。我们发现 I 型 PARPi 接触螺旋 αF 以启动变构链反应，该反应通过多域 PARP-1 分子传播约 40 埃，并最终导致 DNA 结合亲和力增加。螺旋 αF 的结构引导突变破坏了 PARPi 接触并消除了 I 型抑制剂的变构效应，将其转化为非变构 II 型抑制剂。其他破坏 PARP-1 变构的突变，包括在患有 PARPi 的新发耐药卵巢癌患者中发现的一种，也阻止了 I 型 PARPi 在 DNA 断裂时保留 PARP-1。III 型 PARPi 以降低 DNA 结合和促进 DNA 释放的方式影响 PARP-1 变构。促释放（III 型）抑制剂的结构启发修饰将其转化为促保留（I 型）抑制剂，赋予细胞内有效的 PARP-1 捕获并增加其杀死癌细胞的能力。结论我们的研究结果确定了临床 PARPi 对 PARP-1 变构的影响，并证明变构在细胞 PARP-1 捕获中起关键作用，并且可以增加杀死​​癌细胞的效力。结果阐明了微调 PARPi 以实现变构效应并影响 PARP-1 对 DNA 损伤的保留和细胞染色质捕获的分子基础。与癌症相反，其他疾病似乎可以从 PARP-1 抑制中获益，但不能从细胞死亡中获益（例如，心血管疾病、神经退行性疾病和炎症）。我们的研究提供了分子理解和适当的工具集，以创建和评估可调谐 PARPi 的临床应用，其中 PARP-1 捕获和相关的细胞毒性在特定患者中是可取的或不可取的。PARPi 对 PARP-1 变构的影响。PARP-1 (tan) 使用多个域来检测 DNA 断裂，DNA 损伤检测与聚（ADP-核糖）生产变构耦合。PARPi 与催化结构域结合以抑制 PARP-1 活性。I 型 PARPi 影响 PARP-1 变构并在 DNA 上保留 PARP-1（左，UKTT15 为绿色），而 III 型 PARPi 扰乱 PARP-1 变构并从 DNA 中释放 PARP-1（右，veliparib 为红色）。II 型 PARPi 不影响 PARP-1 变构。聚（ADP-核糖）聚合酶-1（PARP-1）抑制剂（PARPi）治疗癌症的成功与它们在DNA断裂位点捕获PARP-1的能力有关。尽管不同形式的 PARPi 都针对酶的催化中心，但它们捕获 PARP-1 的能力各不相同。我们发现几个结构不同的 PARPi 驱动 PARP-1 变构以促进 DNA 断裂的释放。其他抑制剂驱动变构以在 DNA 断裂时保留 PARP-1。此外，我们生成了一种新的 PARPi 化合物，将变构促释放化合物转化为促保留化合物并增加其杀死癌细胞的能力。这些发展与 PARP-1 捕获是可取的或不可取的临床应用相关。