Quantitative High Throughput Screening for Small Molecules Targeting CD47 in Cancer

科学抽象 CD47是一种免疫检查点分子，其下调先天性和适应性抗-CD47抗体的关键方面。 通过肿瘤相关骨髓细胞上的抑制性受体SIRP α的肿瘤免疫应答，包括 巨噬细胞和骨髓源性抑制细胞。CD47在固体中以较高水平表达， 与非恶性组织相比，血液肿瘤与治疗抗性和预后不良相关。 这导致了生物制剂的发展，如阻断SIRP α结合的人源化CD47抗体 目前正在临床试验中测试。不幸的是，毒理学问题，包括贫血有关无处不在 与正常细胞相比，CD47表达和较差的肿瘤微环境（TME）选择性是其临床应用的障碍。 进步。需要额外的SIRP α-CD47阻断方式来实现这一关键的免疫抑制剂的全部潜力。 免疫治疗靶向。 我们假设，使用靶向SIRP α的小分子阻断SIRP α-CD47相互作用将导致SIRP α-CD47相互作用。 更好的TME选择性、更低的毒性、增强的实体瘤渗透，并导致更大的抗肿瘤功效。 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SIRP α靶向 小分子，并取得了实质性的进展，朝着这一目标与我们目前的NIH奖。 与许多免疫检查点一样，SIRP α-CD47是一种蛋白质-蛋白质相互作用复合物， 用我们描述过的小分子靶向。然而，结合X射线晶体学碎片 筛选、蛋白质观察到的异质单量子相干（HSQC）NMR和均匀时间 通过分辨荧光（HTRF），我们鉴定了结合SIRP α的化学型。这些分子 改变了CD47界面上SIRP α区域的构象，提供了一种破坏SIRP α区域的机制。 复杂.我们随后将这些初始X射线筛选命中改进为高度配体效率（LE = 0.45）。 结合SIRP α并抑制CD47相互作用的探针。这些新的SIRP α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，得到 一致的SAR轮廓，并提供一个跳板，以高影响力击中领导运动。使用我们独特 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命中， 选择性的类铅分子。为了支持这一运动，我们建立了一套创新的生物体外 本发明还涉及表征测定以测试它们的抗肿瘤免疫活性和作用机制。 这个MERIT奖延期申请的总体目标是继续开发我们经过验证的 SIRP α靶向命中有效的生物活性先导分子，可根据 具体目标如下： 目标4。有效和选择性的CD47-SIRP α抑制小分子的设计、合成和生化评价 来自验证的SIRP α结合片段命中的分子。这个目标的目标是产生多个候选人 分子通过命中核心的局部优化并生长到相邻的子袋中，具有足够的潜力， 在目标5中确定其生物活性。 目标5。CD47-SIRP α抑制临床相关小分子活性的评价和优化 模型确定小分子SIRP α-CD47抑制剂引发生物学效应所需的效力， 活性，我们将使用一系列经验证的生物活性测定方法评估其体外活性和靶向选择性。 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。 使用充分表征的适应症特异性动物模型进行抗肿瘤免疫活性评价。 我们对这一引人注目的目标的新方法将创造出第一种化学探针来询问 SIRP α-CD47介导的肿瘤细胞杀伤机制。它将为进一步发展一个 针对癌症患者的同类最佳免疫疗法，与其他CD47靶向癌症相比具有显著优势 免疫疗法正在研发中

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.