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

发现用于调节癌症治疗和免疫系统的 SAMHD1 化学探针

• 批准号: 10396629
• 负责人: JAMES T. STIVERS
• 金额: $ 36.71万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10396629
• 关键词: Acyclovir; Antimetabolites; Antineoplastic Agents; Aspartate; Autoimmunity; Binding; Biological; Biological Assay; Biology; Catalytic Domain; Cell Culture Techniques; Cells; Cellular Assay; Cessation of life; Chemicals; Chemistry; Clinical; Custom; Cytarabine; Cytoplasm; DNA Repair; DNA replication fork; Decitabine; Disease; Drug resistance; Enzymes; Exonuclease; Genes; Guanine; Guanosine; Histidine; Homeostasis; Hydrophobicity; Immune; Immune checkpoint inhibitor; Immune system; Immunosuppression; In Vitro; Interferons; Lead; Libraries; Ligands; Malignant Neoplasms; Methods; Molecular; Nucleic Acids; Nucleosides; Nucleotides; Outcome; Pathway interactions; Pharmaceutical Preparations; Pharmacotherapy; Prevention; Proteins; Regulation; Research; Resistance; Retroelements; Role; SAM Domain; Series; Single-Stranded DNA; Site; Specificity; Stimulator of Interferon Genes; Structure; Virus Diseases; anti-cancer; anti-tumor immune response; base; cancer therapy; carcinogenesis; design; dimer; high throughput screening; homologous recombination; immune activation; in vitro Assay; inhibitor; innovation; insight; member; monomer; neoplastic cell; nucleobase; nucleoside analog; repair function; repaired; replication stress; screening; small molecule; small molecule inhibitor; synergism; tool; tripolyphosphate; tumor

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.

无菌α基序结构域-组氨酸天冬氨酸结构域蛋白1（SAMHD 1）是一种酶， 具有dNTP三磷酸水解酶（dNTH）和DNA损伤修复（DDR）的多功能酶 活动越来越清楚的是，酶位于dNTP池之间的关键联系 调节和细胞核酸稳态。癌症治疗的重要性在于它的高度混杂性 dNTR活性是核苷类抗癌药物临床耐药的主要机制 阿糖胞苷和三磷酸地西他滨。磷酸化形式的SAMHD 1（pSAMHD 1）结合到单- 在体外和停滞的复制叉（RF）处的单链DNA。在不存在功能性pSAMHD 1的情况下，肿瘤细胞 与内在复制应激溢出叉相关的ssDNA进入细胞质，触发cGAS/STING 核酸传感途径，从而诱导干扰素刺激的基因。这个新发现的功能 这提出了抑制SAMHD 1可以增强抗肿瘤免疫应答的前景，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剂和激活剂， SAMDH 1活动作为研究工具，将有助于理解SAMHD 1在以下方面的作用： 核苷耐药性和免疫传感途径。这一建议是独一无二的，因为我们利用这两个 高通量筛选（HTS）和片段拴系方法。在目标1中，我们将使用高通量 dNTR检测（Z ′ = 0.87），以筛选霍普金斯大学提供的定制设计的10万成员文库 ChemCore筛选设施。还开发了快速正交二级筛选， 使用一组体外测定和基于细胞的计数器筛选验证和表征其MOA。 预期这些探针靶向SAMHD 1上的多种位点（激活剂位点、催化位点或亚甲基化位点）。 单元接口）。在目标2中，我们的片段拴系方法得到了结构和变构的支持。 四聚体SAMHD 1的激活机制：该酶具有两个紧密相邻的结合口袋 必需的核苷酸激活剂（A1和A2），必须被占据以驱动活性四聚体的形成 单体。靶向A1和A2位点的栓系配体具有最高的促进 SAMHD 1的抑制剂和激活剂的发现，因为这些位点与各种 已知核苷酸根据配体结构产生任一结果。我们已经 鉴定了用于拴系的合适的核苷和小分子片段。在目标3中，一组细胞 将使用测定来阐明验证的探针对（i）细胞dNTP池水平，（ii）RF重新启动， (iii)通过同源重组修复DSB，（iv）增加基于核苷的抗癌药物的效力， 细胞培养中的药物，和（v）预防核苷耐药性。生成的分子应该提供 一组不同的化学探针，有助于我们了解SAMHD 1生物学及其dNTR如何在细胞内表达。 免疫抑制功能可能会增强肿瘤死亡。