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CXCR 4和非典型趋化因子受体ACKR 3在这一过程中起着关键作用。 在免疫反应和器官发育过程中细胞迁移中的作用，通过协调反应， 共享配体CXCL 12。这两种受体都导致许多炎症和自身免疫性疾病， 作为治疗靶点正在积极研究中。然而，目前只有一个FDA批准的 CXCR 4拮抗剂（AMD 3100/Plerixafor），其用途仅限于动员造血干细胞用于骨 骨髓移植，因为它的许多特性是次优的。ACKR 3的治疗靶向是在一个较小的 事实上，大多数已知的化合物是激动剂，并且不清楚如何拮抗CXCR 4。 这个受体。因此，需要靶向CXCR 4和ACKR 3两者的改进的化合物。 作为一种“非典型”受体，人们普遍认为ACKR 3仅通过β-抑制蛋白（而不是G蛋白）发挥作用， 最为人所知的是它能够从细胞外环境中“清除”CXCL 12。通过这样做，ACKR 3 阻止CXCR 4下调并维持其对CXCL 12梯度的反应性。当共表达时 在相同的细胞中，ACKR 3还可以通过异源二聚化、螯合CXCR 4和CXCR 4结合来改变CXCR 4信号传导和运输。 β-抑制蛋白和其他尚未破译的机制。假设ACKR 3在体系结构中绑定CXCL 12， 与CXCR 4相似，在激活后经历相似的构象变化，并共享所有保守的 G蛋白偶联决定簇，其假定的Gi无能是惊人的。更惊人的是， ACKR 3活化对配体和受体修饰的稳健性，而CXCR 4活化被消除 这种变化的微妙之处。由于这种易于激活的性质，大多数非趋化因子（甚至是小的趋化因子）， 分子）配体激活与β-抑制蛋白的ACKR 3缔合，具有未知的下游后果。 尽管这两种受体在疾病中发挥作用，但它们的结构和分子机制仍然存在差异。 单个功能和它们的细胞串扰仍然是难以捉摸的。在这个MPI提案中，Handel和Kufareva 实验室联合收割机分别将他们的实验和计算专业知识与他们对以下方面的深入了解相结合： 趋化因子受体，以解释CXCR 4（Aim 1）和ACKR 3（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（目的1和2），并 了解ACKR 3如何调节CXCR 4的功能（目标3）。为了实现这些目标，具体机制 结合结构（冷冻EM和晶体学）、计算（建模） 和MD）和基于细胞的功能实验，并辅以无偏发现蛋白质组学。这些 研究将提供前所未有的深入了解CXCR 4和ACKR 3的功能，这将产生直接的影响 对小分子疗法的发展，并提供了阻断一个或两个受体的基本原理。 通过揭示一般原则，拟议的研究还将促进对其他问题的理解和针对性。 治疗上重要的趋化因子受体，其被认为是具有挑战性的靶标。

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.