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: 10163140
• 负责人: JAMES T. STIVERS
• 金额: $ 37.46万
• 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/10163140
• 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; Gap Junctions; 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/antagonist; 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.

酶无菌 Alpha 基序结构域-组氨酸天冬氨酸结构域含蛋白 1 (SAMHD1) 是一种 兼具 dNTP 三磷酸水解酶 (dNTPase) 和 DNA 损伤修复 (DDR) 的多功能酶 越来越清楚的是，该酶处于 dNTP 库之间的关键联系。 调节和细胞核酸稳态对癌症治疗的重要意义在于其高度混杂。 dNTPase活性，这是核苷类抗癌药物临床耐药的主要机制 阿糖胞苷和地西他滨三磷酸盐结合单-SAMHD1 (pSAMHD1)。 在缺乏功能性 pSAMHD1 的情况下，肿瘤细胞在体外形成链状 DNA 并处于停滞的复制叉 (RF) 状态。 具有内在复制压力，将叉相关的 ssDNA 溢出到细胞质中，触发 cGAS/STING 核酸传感途径，从而诱导干扰素刺激基因这一新发现的功能。 提出了抑制 SAMHD1 可以增强抗肿瘤免疫反应的前景，特别是在 我们打算发现免疫检查点抑制剂的小分子抑制剂和激活剂。 SAMDH1 活动作为研究工具，有助于了解 SAMHD1 在 核苷耐药性和免疫传感途径是独一无二的，因为我们同时利用了这两种途径。 高通量筛选 (HTS) 和片段束缚方法 在目标 1 中，我们将使用高通量筛选方法。 dNTPase 检测 (Z´ = 0.87) 用于筛选霍普金斯大学提供的定制设计的 100,000 名成员文库 ChemCore 筛选设施也已开发出来，并且将获得成功。 使用一组体外测定和基于细胞的计数器筛选对其 MOA 进行验证和表征。 这些探针预计会针对 SAMHD1 上的多种位点（激活位点、催化位点或子位点）。 在目标 2 中，我们的片段束缚方法受到结构和变构的支持。 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），必须被占据以驱动活性四聚体的形成 靶向 A1 和 A2 位点的束缚配体具有最高的促进潜力。 SAMHD1 抑制剂和激活剂的发现，因为这些位点与各种 已知核苷酸会根据配体结构产生任一结果。 在目标 3 中，确定了用于束缚的适当核苷和小分子片段。 测定将用于阐明经过验证的探针对 (i) 细胞 dNTP 池水平、(ii) RF 重启、 (iii) 通过同源重组进行 DSB 修复，(iv) 提高基于核苷的抗癌药效 在细胞培养中，和药物（v）预防核苷耐药性所产生的分子应提供。 一组多样化的化学探针，有助于我们了解 SAMHD1 生物学及其 dNTPase 的作用 免疫抑制功能可能会促进肿瘤死亡。