Quantitative High Throughput Screening for Small Molecules Targeting CD47 in Cancer

科学摘要 CD47 是一种免疫检查点分子，可下调先天性和适应性抗病毒的关键方面。 通过肿瘤相关骨髓细胞上的抑制性受体 SIRPα 产生肿瘤免疫反应，包括 巨噬细胞和骨髓来源的抑制细胞。 CD47 在固体和 血液肿瘤的发生率高于非恶性组织，并且与治疗耐药性和不良预后相关。 这导致了生物制剂的开发，例如阻断 SIRPα 参与的人源化 CD47 抗体 正在临床试验中进行测试。不幸的是，毒理学问题，包括与普遍存在的贫血相关的 CD47 表达和与正常细胞相比较差的肿瘤微环境 (TME) 选择性是其临床的障碍 进步。需要额外的 SIRPα-CD47 阻断方式来实现这一关键的全部潜力 免疫治疗目标。 我们假设使用靶向 SIRPα 的小分子来阻断 SIRPα-CD47 相互作用将导致 更好的TME选择性，更低的毒性，增强实体瘤的渗透性，并带来更大的抗肿瘤功效。 为了解决这个假设，我们建立了一个创新的多学科项目来开发第一个 SIRPα 靶向 小分子，并在实现这一目标方面取得了实质性进展，并获得了我们当前的 NIH 奖项。 与许多免疫检查点一样，SIRPα-CD47 是一种蛋白质-蛋白质相互作用复合物，难以识别 正如我们所描述的，使用小分子靶向。然而，使用 X 射线晶体学片段的组合 筛选、蛋白质观察异核单量子相干 (HSQC) NMR 和均质时间 通过分辨荧光 (HTRF)，我们鉴定了结合 SIRPα 的化学型。这些分子戏剧性地 改变了 CD47 界面上 SIRPα 区域的构象，提供了一种破坏 复杂。我们随后将这些最初的 X 射线筛选结果改进为高配体效率 (LE = 0.45) 结合 SIRPα 并抑制 CD47 相互作用的探针。这些新颖的 SIRPα 结合分子已被 通过 HTRF、AlphaScreen、等温量热法、HSQC NMR 和 X 射线晶体学验证，得出 一致的搜寻与援救概况，并为领导活动的高影响力打击提供跳板。利用我们独特的 分子设计工具，我们开发了一种基于结构的策略，以进一步优化我们的产品，使其成为有效且有效的产品。 选择性铅样分子。为了支持这项活动，我们建立了一套创新的生物体外实验 表征分析以测试其抗肿瘤免疫活性和作用机制。 此 MERIT 奖励延期申请的总体目标是继续开发我们经过验证的 SIRPα 靶向命中有效的生物活性先导分子，为体内评估做好准备 以下具体目标： 目标 4. 有效且选择性抑制小分子 CD47-SIRPα 的设计、合成和生化评估 来自经过验证的 SIRPα 结合片段命中的分子。该目标的目标是生成多个候选者 通过对命中核心进行局部优化并生长到具有足够效力的相邻子口袋中来形成分子 在目标 5 中确定其生物活性。 目标5. CD47-SIRPα抑制小分子活性在临床相关中的评估和优化 模型。确定小分子 SIRPα-CD47 抑制剂引发生物效应所需的效力 活性，我们将使用一系列经过验证的生物活性来评估它们的体外活性和目标选择性 作用机制测定和肿瘤免疫相互作用表征系统。 这些目标的成功完成将产生用于后续评估的主要候选分子 使用特征明确的适应症特异性动物模型进行抗肿瘤免疫活性评估。 我们针对这一引人注目的目标的新颖方法将创建第一个化学探针来询问 SIRPα-CD47 介导的肿瘤细胞杀伤机制。将为进一步发展打下基础 针对癌症患者的一流免疫疗法，与其他 CD47 靶向癌症相比具有显着优势 免疫疗法目前正在开发中。

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.