Repeated dosing of drugs targeting G protein–coupled receptors can stimulate antagonist tolerance, which reduces their efficacy; thus, strategies to avoid tolerance are needed. The efficacy of AMD3100, a competitive antagonist of the chemokine receptor CXCR4 that mobilizes leukemic blasts from the bone marrow into the blood to sensitize them to chemotherapy, is reduced after prolonged treatment. Tolerance to AMD3100 increases the abundance of CXCR4 on the surface of leukemic blasts, which promotes their rehoming to the bone marrow. AMD3100 inhibits both G protein signaling by CXCR4 and β-arrestin1/2–dependent receptor endocytosis. We demonstrated that biased antagonists of G protein–dependent chemotaxis but not β-arrestin1/2 recruitment and subsequent receptor endocytosis avoided tolerance. The peptide antagonist X4-2-6, which is derived from transmembrane helix 2 and extracellular loop 1 of CXCR4, limited chemotaxis and signaling but did not promote CXCR4 accumulation on the cell surface or cause tolerance. The activity of X4-2-6 was due to its distinct mechanism of inhibition of CXCR4. The peptide formed a ternary complex with the receptor and its ligand, the chemokine CXCL12. Within this complex, X4-2-6 released the portion of CXCL12 critical for receptor-mediated activation of G proteins but enabled the rest of the chemokine to recruit β-arrestins to the receptor. In contrast, AMD3100 displaced all components of the chemokine responsible for CXCR4 activation. We further identified a small molecule with similar biased antagonist properties to those of X4-2-6, which may provide a viable alternative to patients when antagonist tolerance prevents drugs from reaching efficacy.

重复给药靶向G蛋白偶联受体的药物会刺激拮抗剂的耐受性，从而降低其疗效。因此，需要避免宽容的策略。长期治疗后，AMD3100（趋化因子受体CXCR4的竞争性拮抗剂，将白血病细胞从骨髓中转移到血液中使其对化疗敏感）的功效降低。对AMD3100的耐受性增加了白血病母细胞表面CXCR4的丰度，从而促进了它们向骨髓的归巢。AMD3100抑制CXCR4和β-arrestin1/ 2依赖性受体胞吞作用的G蛋白信号传导。我们证明了偏倚的G蛋白依赖性趋化性拮抗剂而不是β-arrestin1/ 2募集以及随后的受体胞吞作用避免了耐受性。肽拮抗剂X4-2-6，它来自CXCR4的跨膜螺旋2和细胞外环1，限制了趋化性和信号传导，但不促进CXCR4在细胞表面的积累或引起耐受。X4-2-6的活性是由于其抑制CXCR4的独特机制。该肽与受体及其配体趋化因子CXCL12形成三元复合物。在该复合物中，X4-2-6释放了CXCL12对受体介导的G蛋白活化至关重要的部分，但使其余的趋化因子使β-arrestin募集到受体上。相反，AMD3100取代了负责CXCR4激活的趋化因子的所有组件。我们进一步确定了一种具有与X4-2-6相似的偏向拮抗剂特性的小分子，