Discovery of Chemical Probes of SAMHD1 for Modulation of Cancer Therapy and the Immune System

酶不育α基序结构域-组氨酸天冬氨酸结构域包含蛋白1(SAMHD1)是一种 同时具有dNTP三磷酸水解酶(DNTPase)和DNA损伤修复(DDR)的多功能酶 活动。越来越清楚的是，这种酶位于dNTP池之间的关键连接点 调控与细胞核酸动态平衡。对癌症治疗有重要意义的是它的高度混杂 DNTPase活性是临床抗癌药物耐药的主要机制 阿糖胞苷和三磷酸地西他滨。一种磷酸化形式的SAMHD1(PSAMHD1)与单个- 在体外和停滞的复制叉处(RF)搁浅的DNA。在缺乏功能pSAMHD1的情况下，肿瘤细胞 在内在复制压力下，叉状相关的单链DNA溢出到细胞质中，触发cGAS/STIN 核酸感应通路，从而诱导干扰素刺激的基因。这个新发现的功能 提出了抑制SAMHD1可以增强抗肿瘤免疫反应的前景，特别是在 免疫检查点抑制剂的背景。我们打算发现小分子抑制物和激活剂 作为研究工具的SAMDH1活动，将有助于理解SAMHD1在 核苷耐药和免疫传感通路。这项建议是独一无二的，因为我们既利用了 高通量筛选(HTS)和片段系留方法。在目标1中，我们将使用高吞吐量 DNTPase试验(Z？=0.87)以筛选可在霍普金斯大学获得的定制设计的100,000人文库 ChemCore筛查设施。还开发了快速正交二次筛选，并将点击 使用一组体外分析和基于细胞的计数器筛选来验证和表征他们的MOA。 这些探针预计将针对SAMHD1上的各种位点(激活部位、催化部位或亚基 单元接口)。在目标2中，我们的片段系链方法是由结构和变构支持的 四聚体SAMHD1的激活机制：该酶具有紧密相邻的两个结合口袋 必需的核苷酸激活剂(A1和A2)，它们必须被占据以驱动活性四聚体的形成 来自单体。靶向A1和A2位点的系链配体具有最高的促进作用 SAMHD1抑制剂和激活剂的发现，因为这些位点与不同的 根据配体结构的不同，核苷酸已经知道会导致这两种结果中的任何一种。我们已经这么做了 确定了合适的核苷和小分子片段用于系留。在《目标3》中，一组细胞 检测将被用来阐明验证的探针对(I)细胞dNTP池水平，(Ii)RF重新启动， (3)通过同源重组修复DSB；(4)提高抗癌核苷类化合物的效力 细胞培养中的药物，以及(V)防止核苷耐药性。由此产生的分子应该会提供 一组不同的化学探针，有助于我们了解SAMHD1生物学及其dNTPase如何 免疫抑制功能可能会增加肿瘤的死亡率。

The enzyme Sterile Alpha Motif domain-Histidine aspartate Domain-containing protein 1 (SAMHD1) is a multifunctional enzyme possessing both dNTP triphosphohydrolase (dNTPase) and DNA damage repair (DDR) activities. It is becoming increasingly clear that the enzyme lies at a critical nexus between dNTP pool regulation and cellular nucleic acid homeostasis. Of significance to cancer therapy is its highly promiscuous dNTPase activity, which is the primary mechanism of clinical resistance to the nucleoside anticancer drugs cytarabine and decitabine triphosphate. A phosphorylated form of SAMHD1 (pSAMHD1) binds to single- stranded DNA in vitro and at stalled replication forks (RF). In the absence of functional pSAMHD1, tumor cells with intrinsic replication stress spill fork-associated ssDNA into the cytoplasm, triggering the cGAS/STING nucleic acid-sensing pathways, thereby inducing interferon-stimulated genes. This newly discovered function raises the prospect that inhibition of SAMHD1 could enhance anti-tumor immune responses, particularly in the context of immune checkpoint inhibitors. We intend to discover small molecule inhibitors and activators of SAMDH1 activities to serve as research tools that will facilitate understanding of the role of SAMHD1 in nucleoside drug resistance and immune sensing pathways. This proposal is unique because we utilize both high-throughput screening (HTS) and fragment tethering approaches. In Aim 1 we will use a high-throughput dNTPase assay (Z´ = 0.87) to screen a custom-designed 100,000-member library available at the Hopkins ChemCore screening facility. Rapid orthogonal secondary screens have also been developed and hits will be validated and characterized for their MOA using a panel of in vitro assays and cell-based counter screens. These probes are expected to target a diversity of sites on SAMHD1 (activator sites, the catalytic site, or sub- unit interfaces). In Aim 2, our fragment tethering approach is supported by the structure and allosteric activation mechanism of tetrameric SAMHD1: the enzyme has closely adjacent binding pockets for two essential nucleotide activators (A1 and A2), which must be occupied to drive formation of the active tetramer from monomers. Tethered ligands that target the A1 and A2 sites have the highest potential to facilitate discovery of both inhibitors and activators of SAMHD1 because co-occupancy of these sites with various nucleotides is already known to give rise to either outcome depending on the ligand structure. We have already identified appropriate nucleoside and small molecule fragments for tethering. In Aim 3, a panel of cellular assays will be used to elucidate the effects of validated probes on (i) cellular dNTP pool levels, (ii) RF restart, (iii) DSB repair via homologous recombination, (iv) increasing the potency of anticancer nucleoside-based drugs in cell culture, and (v) prevention of nucleoside drug resistance. The resulting molecules should provide a diverse set of chemical probes that facilitate our understanding of SAMHD1 biology and how its dNTPase and immune suppression functions might enhance tumor death.