Virginia Commonwealth University School of Medicine, Richmond, VA, United States; PAKA Pulmonary Pharmaceuticals, Acton, MA, United States.
Vitam Horm. 2017;105:19-37. doi: 10.1016/bs.vh.2017.03.004. Epub 2017 May 18.
Amgen solved the high-resolution cocrystal structure of erythropoietin (EPO) bound to the extracellular part of the receptor (EPOR) in 1998, which reveals that the EPO-EPOR interaction surface is formed by 11 salt bridges, 17 H-bonds, and 2 hydrophobic clusters centered at a pair of crucial phenylalanines (F93). The EPOR has two domains, one that penetrates the membrane and a second extracellular domain that forms one arm of the binding site for the EPO ligand. The complete competent receptor-binding site is a homodimer of EPOR with the two arms forming a funnel-shaped cup where EPO binds. The two binding arms of the EPOR dimer meet at the membrane at a 120 degree angle, which Amgen characterizes as, "erythropoietin imposes a unique angular relationship and orientation that is responsible for optimal signaling." They come to this conclusion, because the EPOR cocrystallized with 2 equivalents of a 20 residue EPO mimetic peptide created at Robert Wood Johnson (RWJ) activates the receptor with a 3 order of magnitude reduction in potency, and the binding arms are forced to meet at the membrane with an angle of 180 degrees. The vast interaction surface between EPO and EPOR forms a singularly important three-dimensional structure responsible for hematopoietic stem cell proliferation and differentiation-this is Amgen's conclusion. This goal of this work is to present experimental and computational evidence that the Amgen structure is a postsignaling off-state and that the RWJ structure with the partially active peptide mimetics is an on-state. A detailed side-by-side comparison of the two structures will be presented along with literature evidence that calls into question the Amgen claim that their structure is a unique on-state. A computational fragment-based drug discovery method applied to the RWJ structure was used to locate and characterize a new predicted small molecule binding site and a fragment analysis was performed based on theories of asymmetry to create a proposed agonist with MW<300. When this molecule was experimentally tested, it displaced radiolabeled EPO with nanomolar potency and transformed human hematopoietic stem cells into red blood cells with subnanomolar potency. Obviously, this small molecule makes none of the EPO-EPOR interactions that Amgen stated were essential for fully turning on the receptor and provides strong evidence that stabilizing receptor asymmetry, not specific interactions, is the critical factor needed for activating signal transduction. Finally, when the agonist was altered to remove the asymmetric component, it still was able to displace radiolabeled EPO in competition binding experiments, but it no longer activated the receptor.
安进公司于 1998 年解决了促红细胞生成素（EPO）与受体（EPOR）胞外部分结合的高分辨率共晶结构问题，揭示了 EPO-EPOR 相互作用表面由 11 个盐桥、17 个氢键和 2 个以一对关键苯丙氨酸（F93）为中心的疏水区组成。EPOR 有两个结构域，一个穿透膜，另一个是胞外结构域，形成 EPO 配体结合位点的一个臂。完整的有效受体结合位点是 EPOR 的同源二聚体，两个臂形成一个漏斗形杯，EPO 结合于此。EPOR 二聚体的两个结合臂在膜上以 120 度的角度相遇，安进公司将其描述为，“促红细胞生成素施加了独特的角度关系和取向，这是最佳信号传递的原因。”他们得出这个结论是因为与 2 当量的罗伯特·伍德·约翰逊（RWJ）设计的 20 残基 EPO 模拟肽共结晶的 EPOR 以 3 个数量级的效力降低激活受体，并且结合臂被迫以 180 度的角度在膜上相遇。EPO 和 EPOR 之间的巨大相互作用表面形成了一个单一的重要三维结构，负责造血干细胞的增殖和分化——这是安进公司的结论。这项工作的目标是提供实验和计算证据，证明安进公司的结构是一种信号后关闭状态，而 RWJ 结构与部分活性肽模拟物是一种开启状态。将对这两种结构进行详细的并排比较，并结合文献证据，对安进公司声称其结构是独特的开启状态的说法提出质疑。将应用于 RWJ 结构的基于片段的药物发现方法用于定位和表征一个新的预测小分子结合位点，并根据不对称理论进行片段分析，以创建一个具有 MW<300 的拟议激动剂。当这种分子进行实验测试时，它以纳摩尔效力置换放射性标记的 EPO，并以亚纳摩尔效力将人造血干细胞转化为红细胞。显然，这种小分子不会产生安进公司所说的对完全激活受体至关重要的任何 EPO-EPOR 相互作用，并提供了强有力的证据，证明稳定受体不对称性而不是特定相互作用是激活信号转导所必需的关键因素。最后，当激动剂被改变以去除不对称成分时，它仍然能够在竞争结合实验中置换放射性标记的 EPO，但它不再激活受体。
