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: 10650716
• 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 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10650716
• 关键词: Acyclovir; Antimetabolites; Antineoplastic Agents; Aspartate; Autoimmune Diseases; 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; 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; Phosphorylation; 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; 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.

酶无菌α基序域 - 希斯丁定蛋白天冬氨酸蛋白1（SAMHD1）是一个 具有DNTP三磷酶（DNTPase）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池之间的关键下一个 调节和细胞核酸稳态。对癌症治疗的重要性是其高度混杂的 DNTPase活性，这是对核横向抗癌药物临床抗性的主要机制 细胞押滨和三磷酸决定性磷酸盐。 SAMHD1（PSAMHD1）的磷酸化形式与单个 滞留的DNA体外和停滞的复制叉（RF）。在没有功能性PSAMHD1的情况下，肿瘤细胞 固有的复制应力溢出叉相关的ssDNA进入细胞质，触发CGA/sting 核酸 - 敏感性途径，从而诱导干扰刺激的基因。这个新发现的功能 提高了抑制SAMHD1可以增强抗肿瘤免疫调查的前景，特别是在 免疫检查点抑制剂的上下文。我们打算发现小分子抑制剂和激活剂 SAMDH1活动是作为研究工具的活动，以促进了解SAMHD1在 核苷耐药性和免疫传感途径。该建议是独一无二的 高通量筛选（HTS）和碎片绑扎方法。在AIM 1中，我们将使用高通量 DNTPase分析（Z´= 0.87）筛选霍普金斯的自定义设计的100,000名成员的库 ChemCore筛选设施。还已经开发了快速的正交次级屏幕，命中将是 使用一系列体外测定和基于细胞的计数器筛网对其MOA进行了验证和特征。 这些问题有望针对SAMHD1（激活剂位点，催化位点或亚下）的各种位点。 单位接口）。在AIM 2中，我们的碎片束缚方法得到了结构和变构的支持 四聚体SAMHD1的激活机制：酶具有两个酶的结合口袋的两个 必需的核苷酸激活剂（A1和A2），必须占用其驱动活性四聚体的形成 来自单体。靶向A1和A2位点的束缚配体具有促进的最高潜力 发现SAMHD1的抑制剂和激活剂，因为这些位点的共同占领 众所周知，核苷酸已取决于配体结构。我们已经 确定了适当的核侧向和小分子片段以束缚。在AIM 3中，一组蜂窝 测定将用于阐明验证问题对（i）细胞DNTP池级的影响，（ii）RF重新启动， （iii）通过同源重组的DSB修复，（iv）增加抗癌基于核核的效力 细胞培养中的药物，（v）预防核苷耐药性。由此产生的分子应提供 一组各种化学问题，促进我们对SAMHD1生物学的理解以及其DNTPase如何 免疫抑制功能可能会增强肿瘤死亡。