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.

无菌α基序结构域-组氨酸天冬氨酸结构域-含蛋白1 （SAMHD1）是一种酶