Quantitative High Throughput Screening for Small Molecules Targeting CD47 in Cancer

定量高通量筛选癌症中靶向 CD47 的小分子

• 批准号: 10557909
• 负责人: Thomas W Miller
• 金额: $ 26.46万
• 依托单位: INSTITUT JEAN PAOLI & IRENE CALMETTES CENTRE REGIONAL DE LUTTE CONTRE LE CANCER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10557909
• 关键词: Address; Adverse event; Affinity; Anemia; Animal Model; Antibodies; Antigen Presentation; Antisense Oligonucleotides; Antitumor Response; Award; Binding; Biochemical; Biological; Biological Assay; Biological Products; Biophysics; Blocking Antibodies; CD47 gene; CD47-SIRPα; Calorimetry; Cancer Biology; Cancer Patient; Catalogs; Cell surface; Cells; Characteristics; Chemicals; Chemosensitization; Clinical; Clinical Trials; Collaborations; Complex; Cytotoxic T-Lymphocytes; Data; Detection; Development; Down-Regulation; Drug Kinetics; Evaluation; Exhibits; Fluorescence; Foundations; Goals; Hematologic Neoplasms; Hybrids; Immune; Immune Evasion; Immunotherapy; In Vitro; Interferometry; Lead; Libraries; Ligand Binding; Ligands; Macrophage; Malignant Neoplasms; Measures; Mediating; Modality; Modeling; Molecular; Molecular Conformation; Monoclonal Antibodies; Myeloid Cells; Myeloid-derived suppressor cells; National Center for Advancing Translational Sciences; Non-Malignant; Normal Cell; PTPNS1 gene; Pathway interactions; Penetration; Phagocytosis; Pharmaceutical Preparations; Pre-Clinical Model; Prognosis; Property; Protein Isoforms; Proteins; Recombinants; Research; Robotics; Roentgen Rays; Scanning; Self Tolerance; Series; Signal Transduction; Solid Neoplasm; Structure; Structure-Activity Relationship; System; T-Lymphocyte; Testing; Therapeutic; Thermodynamics; Time; Tissues; Toxic effect; Toxicology; United States National Institutes of Health; X-Ray Crystallography; anti-tumor immune response; biophysical properties; cancer immunotherapy; cancer type; cell killing; clinically relevant; counterscreen; cytotoxic; cytotoxicity; design; experience; high throughput screening; immune checkpoint; improved; in silico; in vitro activity; in vivo evaluation; inhibitor; innovation; lead candidate; lead optimization; multidisciplinary; neoplastic cell; novel; novel strategies; pre-clinical; programs; protein protein interaction; quantum; receptor; research clinical testing; screening; small molecule; small molecule libraries; therapy design; therapy resistant; tool; tumor; tumor microenvironment; virtual library; virtual screening; Address; Adverse event; Affinity; Anemia; Animal Model; Antibodies; Antigen Presentation; Antisense Oligonucleotides; Antitumor Response; Award; Binding; Biochemical; Biological; Biological Assay; Biological Products; Biophysics; Blocking Antibodies; CD47 gene; CD47-SIRPα; Calorimetry; Cancer Biology; Cancer Patient; Catalogs; Cell surface; Cells; Characteristics; Chemicals; Chemosensitization; Clinical; Clinical Trials; Collaborations; Complex; Cytotoxic T-Lymphocytes; Data; Detection; Development; Down-Regulation; Drug Kinetics; Evaluation; Exhibits; Fluorescence; Foundations; Goals; Hematologic Neoplasms; Hybrids; Immune; Immune Evasion; Immunotherapy; In Vitro; Interferometry; Lead; Libraries; Ligand Binding; Ligands; Macrophage; Malignant Neoplasms; Measures; Mediating; Modality; Modeling; Molecular; Molecular Conformation; Monoclonal Antibodies; Myeloid Cells; Myeloid-derived suppressor cells; National Center for Advancing Translational Sciences; Non-Malignant; Normal Cell; PTPNS1 gene; Pathway interactions; Penetration; Phagocytosis; Pharmaceutical Preparations; Pre-Clinical Model; Prognosis; Property; Protein Isoforms; Proteins; Recombinants; Research; Robotics; Roentgen Rays; Scanning; Self Tolerance; Series; Signal Transduction; Solid Neoplasm; Structure; Structure-Activity Relationship; System; T-Lymphocyte; Testing; Therapeutic; Thermodynamics; Time; Tissues; Toxic effect; Toxicology; United States National Institutes of Health; X-Ray Crystallography; anti-tumor immune response; biophysical properties; cancer immunotherapy; cancer type; cell killing; clinically relevant; counterscreen; cytotoxic; cytotoxicity; design; experience; high throughput screening; immune checkpoint; improved; in silico; in vitro activity; in vivo evaluation; inhibitor; innovation; lead candidate; lead optimization; multidisciplinary; neoplastic cell; novel; novel strategies; pre-clinical; programs; protein protein interaction; quantum; receptor; research clinical testing; screening; small molecule; small molecule libraries; therapy design; therapy resistant; tool; tumor; tumor microenvironment; virtual library; virtual screening

SCIENTIFIC ABSTRACT CD47 is an immune checkpoint molecule that downregulates key aspects of both the innate and adaptive anti- tumor immune response via the inhibitory receptor SIRPα on tumor associated myeloid cells including macrophage and myeloid-derived suppressor cells. CD47 is expressed at higher levels in solid and hematological tumors than nonmalignant tissues and correlates with treatment resistance and poor prognosis. This has led to the development of biologics such as humanized CD47 antibodies that block SIRPα engagement which are being tested in clinical trials. Unfortunately, toxicological issues, including anemia related to ubiquitous CD47 expression and poor tumor microenvironment (TME) selectivity vs normal cells, are barriers to their clinical advancement. Additional SIRPα-CD47 blocking modalities are needed to realize the full potential of this critical immunotherapy target. We hypothesize that using small molecules targeting SIRPα to block SIRPα-CD47 interaction will result in better TME selectivity, lower toxicity, enhanced solid tumor penetration, and lead to greater anti-tumor efficacy. To address this hypothesis, we built an innovative multidisciplinary program to develop the first SIRPα-targeting small molecules and made substantial progress toward this goal with our current NIH award. Like many immune checkpoints, SIRPα-CD47 is a protein-protein interaction complex and is challenging to target using small molecules as we described. However, using a combination of X-ray crystallography fragment screening, protein-observed Heteronuclear Single Quantum Coherence (HSQC) NMR, and Homogeneous Time Resolved Fluorescence (HTRF), we identified chemotypes that bound SIRPα. These molecules dramatically altered the conformation of SIRPα regions at the CD47 interface, providing a mechanism for disruption of the complex. We subsequently improved these initial X-ray screening hits into highly ligand-efficient (LE = 0.45) probes that bound SIRPα and inhibited CD47 interaction. These novel SIRPα-binding molecules have been validated by HTRF, AlphaScreen, isothermal calorimetry, HSQC NMR, and X-ray crystallography, yielding a consistent SAR profile and provide a springboard to a high impact hit to lead campaign. Using our unique molecular design tools, we developed a structure-based strategy to further optimize our hits into potent and selective lead-like molecules. To support this campaign, we established an innovative set of biological in vitro characterization assays to test their anti-tumor immune activity and mechanism of action. The overall objective of this MERIT award extension application is to continue the development of our validated SIRPα-targeting hits into potent biologically active lead molecules ready for in vivo evaluation according to the following specific aims: Aim 4. Design, synthesis, and biochemical evaluation of potent and selective CD47-SIRPα inhibiting small molecules from validated SIRPα-bound fragment hits. The goal of this aim is to generate multiple candidate molecules by local optimization of the hit core and growing into adjacent subpockets with sufficient potency to establish their biological activity in Aim 5. Aim 5. Evaluation and optimization of CD47-SIRPα inhibiting small molecule activities in clinically-relevant models. To determine the potency necessary for small molecule SIRPα-CD47 inhibitors to elicit biological activity, we will evaluate their in vitro activity and target selectivity using a series of validated biological mechanism of action assays and tumor-immune interaction characterization systems. The successful completion of these aims will result in lead candidate molecules for subsequent evaluation of anti-tumor immune activity evaluation using well-characterized indication-specific animal models. Our novel approach to this compelling target will create first of their kind chemical probes to interrogate the mechanisms of SIRPα-CD47-mediated tumor cell killing. It will lay the foundation for further development of a best-in-class immunotherapy for cancer patients with significant advantages over other CD47-targeted cancer immunotherapies now in development.

科学摘要 CD47是一种免疫切口分子，它下调了先天和适应性抗的关键方面 通过抑制性受体SIRPα对肿瘤相关的髓样细胞的肿瘤免疫反应，包括 巨噬细胞和髓样衍生的抑制细胞。 CD47在固体和 血液学肿瘤比非恶性组织，与治疗耐药性和预后不良相关。 这导致了生物制剂的发展，例如人源化CD47抗体，这些抗体阻断了SIRPα参与度 在临床试验中进行了测试。不幸的是，毒理学问题，包括与无处不在的贫血 CD47表达和较差的肿瘤微环境（TME）选择性与正常细胞是其临床的障碍 进步。需要其他SIRPα-CD47阻塞方式来实现此关键的全部潜力 免疫疗法靶标。 我们假设使用靶向SIRPα的小分子阻断SIRPα-CD47相互作用将导致 更好的TME选择性，较低的毒性，增强实体瘤的渗透率，并提高抗肿瘤效率。 为了解决这一假设，我们建立了一个创新的多学科计划，以开发第一个sirpα靶向 小分子并通过我们目前的NIH奖取得了实质性的进步。 像许多免疫粘点一样，SIRPα-CD47是一种蛋白质 - 蛋白质相互作用复合物，对 如我们所述，使用小分子的靶标。但是，使用X射线晶体学片段的组合 筛选，蛋白质观察的异核单量子相干性（HSQC）NMR和均匀时间 已解决的荧光（HTRF），我们确定了结合SIRPα的化学型。这些分子急剧 改变了CD47界面处SIRPα区域的构象，提供了破坏的机制 复杂的。随后，我们将这些初始X射线筛选命中改进了高度配体效率（LE = 0.45） 结合SIRPα并抑制CD47相互作用的问题。这些新型的SIRPα结合分子已经 由HTRF，Alphascreen，等温量热法，HSQC NMR和X射线晶体学验证，得出 一致的SAR概况，并为领先运动带来了高影响力的跳板。使用我们的独特 分子设计工具，我们制定了一种基于结构的策略，以进一步优化潜在的击球和 选择性铅样分子。为了支持这项运动，我们建立了一套创新的体外生物学 表征测定法测试其抗肿瘤免疫活性和作用机理。 该优异奖扩展申请的总体目标是继续开发我们经过验证的 靶向SIRPα靶向击中潜在的生物活性铅分子 遵循特定目的： AIM 4。对电势和选择性CD47-SIRPα的设计，合成和生化评估抑制了小 来自经过验证的SIRPα结合片段命中的分子。该目标的目的是产生多个候选人 分子通过局部优化命中芯，并成长为相邻的子渠，并具有足够的效力 在目标5中建立生物学活性。 AIM 5。抑制与临床相关的小分子活性的CD47-SIRPα的评估和优化 型号。确定小分子SIRPα-CD47抑制剂的必要效力以引起生物学 活动，我们将使用一系列经过验证的生物学评估其体外活性和靶向选择性 作用评估机理和肿瘤免疫相互作用表征系统。 这些目标的成功完成将导致铅候选分子，以随后评估 使用良好的特异性动物模型，抗肿瘤免疫活性评估。 我们实现这一引人入胜的目标的新颖方法将首先创建他们的化学问题来审问 SIRPα-CD47介导的肿瘤细胞杀死的机制。它将为进一步发展的基础 与其他针对CD47的癌症相比，具有显着优势的癌症患者的一流免疫疗法 现在正在开发免疫疗法。