It's a tug of war: structure, consequences, and inhibition of CXCR4 and ACKR3 responses to lymphocyte chemoattractant CXCL12

G蛋白偶联趋化因子受体CXCR4和非典型趋化因子受体ACKR3起关键作用 在免疫反应和器官发育中的细胞迁移中的作用，通过对 共享配体，CXCL12。这两种受体都会导致许多炎症性和自身免疫性疾病 作为治疗靶点，正在积极研究中。然而，目前只有一家FDA批准 CXCR4拮抗剂(AMD3100/Plerixafor)，其用途仅限于为骨动员造血干细胞 骨髓移植，因为它的许多特性都不是最理想的。ACKR3的治疗靶向性较低 CXCR4比CXCR4处于成熟阶段；事实上，大多数已知的化合物都是激动剂，目前还不清楚如何拮抗 这个感受器。因此，需要针对CXCR4和ACKR3的改进化合物。 作为一种“非典型”受体，ACKR3被广泛认为只通过β-arrestin(而不是G蛋白)发挥作用。 最广为人知的是它能从细胞外环境中“清除”CXCL12。通过这样做，ACKR3 防止CXCR4下调，并保持其对CXCL12梯度的响应。当共同表达时 在同一细胞中，ACKR3还可以通过异源二聚、隔离 β-arrestin和其他尚未破译的机制。假设ACKR3在体系结构中绑定了CXCL12 类似于CXCR4，在激活时经历类似的构象变化，并分享所有保守的 G蛋白偶联决定因素，其推定的胃肠道功能不全是惊人的。更令人震惊的是， ACKR3激活对配体和受体修饰的稳健性，而CXCR4激活被取消 这种变化中最微妙的一种。由于这种易于激活的性质，大多数非趋化因子(甚至是很小的 分子)配体激活ACKR3与β-arrestin的结合，下游后果未知。 尽管这两种受体在疾病中发挥了作用，但其背后的结构和分子机制 个体功能及其细胞串扰仍然难以捉摸。在这份MPI提案中，亨德尔和库法列娃 实验室分别将他们的实验和计算专业知识与他们对 趋化因子受体，以解释CXCR4(Aim 1)和ACKR3(Aim 2)的不同激活机制 结构和动力学的观点，了解如何抑制这些受体(目标1和2)，并 了解ACKR3如何调节CXCR4的功能(目标3)。为了实现这些目标，具体的机制 通过结构(冷冻-EM和结晶学)、计算(建模)相结合的方法来探索假设 和MD)和基于细胞的功能实验，并辅之以无偏见的发现蛋白质组学。这些 研究将对CXCR4和ACKR3的功能提供前所未有的见解，这将产生直接影响 关于小分子疗法的发展，并提供了阻断一个或两个受体的理由。 通过揭示一般原则，拟议的研究也将促进对其他 具有重要治疗意义的趋化因子受体，被认为是具有挑战性的靶点。

The G protein-coupled chemokine receptor, CXCR4, and the atypical chemokine receptor, ACKR3, play critical roles in cell migration during immune responses and organ development, through coordinated responses to a shared ligand, CXCL12. Both receptors contribute to numerous inflammatory and autoimmune diseases and are under active investigation as therapeutic targets. Nevertheless, there is currently only one FDA-approved CXCR4 antagonist (AMD3100/Plerixafor), and its use is limited to mobilizing hematopoietic stem cells for bone marrow transplants, because many of its properties are suboptimal. Therapeutic targeting of ACKR3 is at a less mature stage than CXCR4; in fact, most known compounds are agonists, and it is unclear how to antagonize this receptor. Improved compounds targeting both CXCR4 and ACKR3 are therefore needed. As an "atypical" receptor, ACKR3 is widely assumed to function only through β-arrestin (and not G proteins), and is best known for its ability to “scavenge” CXCL12 from the extracellular environment. By doing so, ACKR3 prevents downregulation of CXCR4 and maintains its responsiveness to CXCL12 gradients. When co-expressed in the same cell, ACKR3 can also alter CXCR4 signaling and trafficking via heterodimerization, sequestration of β-arrestin, and other as-yet-undeciphered mechanisms. Given that ACKR3 binds CXCL12 in an architecture similar to CXCR4, undergoes similar conformational changes upon activation, and shares all of the conserved G protein-coupling determinants, its presumed Gi incompetency is striking. Even more striking is the exceptional robustness of ACKR3 activation to ligand and receptor modifications, whereas CXCR4 activation is abrogated by the subtlest of such changes. Because of this activation-prone nature, most non-chemokine (and even small molecule) ligands activate ACKR3 association with β-arrestin, with unknown downstream consequences. Despite the role of the two receptors in disease, the structural and molecular mechanisms underlying their individual functions and their cellular crosstalk remain elusive. In this MPI proposal, the Handel and Kufareva labs combine their experimental and computational expertise, respectively, with their in-depth knowledge of chemokine receptors, to explain the distinct activation mechanisms of CXCR4 (Aim 1) and ACKR3 (Aim 2) from the standpoint of structure and dynamics, to understand how to inhibit these receptors (Aims 1 and 2), and to understand how ACKR3 regulates the function of CXCR4 (Aim 3). To achieve these aims, specific mechanistic hypotheses are probed with a combination of structural (cryo-EM and crystallography), computational (modeling and MD) and cell-based functional experiments, and complemented by unbiased discovery proteomics. These studies will deliver unprecedented insight into the function of CXCR4 and ACKR3, which will have a direct impact on the development of small molecule therapeutics and provide the rationale for blocking one or both receptors. By revealing general principles, the proposed studies will also advance the understanding and targeting of other therapeutically important chemokine receptors, which are considered challenging targets.