Identification of a small molecule inhibitor of the IL-2/IL-2R alpha receptor interaction which binds to IL-2
Identification of a small molecule inhibitor of the IL-2/IL-2R alpha receptor interaction which binds to IL-2
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DOI：
10.1021/ja970702x
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发表时间：
1997-08-13
影响因子：
15
通讯作者：
Ju, G
中科院分区：
文献类型：
--
作者：
Tilley, JW;Chen, L;Ju, G
白细胞介素-2 (IL-2) 是一种 15.5 kDa 的细胞因子，在活化 T 细胞的生长中起主要作用。 IL-2 通过与由 R、和 γ 链组成的异三聚体受体复合物以皮摩尔亲和力结合在 T 细胞表面，从而刺激 T 细胞增殖。 1 识别 R 受体亚基 (IL-2RR) 并阻断 IL-2 结合的抗体已被证明作为免疫抑制剂在临床上有效，1, 2 因此，我们寻找能够阻断 IL-2/IL-2RR 相互作用的小分子作为抗体药物的潜在口服活性后继者。此类药物的设计基于通过 IL-2 的 X 射线晶体学3、4 和 NMR 研究 5 获得的结构信息的组合，这些信息揭示了主链包括一束四个 R 螺旋（A、B、C 和 D），具有上下排列和人 IL-2 的定点诱变，确定了 AB 环中的残基（K35、R38、T41、F42、K43、Y45）和B 螺旋（E62、L72）对于 IL-2 与 IL-2RR 的结合至关重要。 6, 7 旨在模拟 IL-2 R38-F42 区域的一系列酰基苯丙氨酸衍生物中的一个成员被发现是 IL-2/IL-2RR 结合的竞争性抑制剂，IC50 为中微摩尔。结构-活性研究合成了 1，IC50 为 3 µM。尽管酰基苯丙氨酸衍生物被设计为通过模拟IL-2配体的残基R38和F42来与IL-2RR复合，但我们考虑了它们可能与配体相互作用的可能性。使用统一 15N 标记的 IL-2 进行的 NMR 研究表明，先前通过定点诱变鉴定的结合表位附近残基的共振以浓度依赖性方式选择性地受到 1 的干扰，但不受其无活性对映体 2 的干扰，表明 1 通过与 IL-2 结合来抑制 IL-2/IL-2RR 结合。据我们所知，这代表了细胞因子/细胞因子受体相互作用的小分子非肽抑制剂的第一个充分表征的例子。使用闪烁邻近竞争性结合测定评估了化合物抑制 IL-2/IL-2RR 相互作用的能力。 8 该测定主要测量在不同浓度的抑制剂化合物存在下 125 I 标记的 IL-2 与固定的可溶性 IL-2RR 的结合。根据抑制曲线 Hill 图确定，化合物 1 在 pH 7.4 时抑制 IL-2 与 IL-2RR 的结合，IC50 为 3 µM。相比之下，它的对映体 2 在测试的最高浓度 (500 µM) 下仅产生 14% 的结合抑制，而 IL-2 的 IC50 为 13 nM。为了表征1的抑制活性，在存在不同浓度的1或未标记的IL-2的情况下，用不同浓度的125I标记的IL-2进行结合测定。进行了 Scatchard 分析，并将每个抑制剂浓度下的 [125I] IL-2 结合的 Kd 值与抑制剂浓度绘制在 Schild 图 9 中（图 1），这表明观察到的 1 的抑制活性与竞争性可逆抑制一致。为了评估抑制机制，使用均匀富集 15N 的 IL-2 进行 NMR 实验。 10 通过分析 3D 1H-1H-15N-TOCSY-和 NOESY-HSQC 谱图获得 1H-15N NMR 归属。 11 在大多数情况下，这些共振分配被确定为与之前发表的类似。 12 1H-15N 化学位移的扰动是在 2D 1H-15N-HSQC 光谱中测量的，该光谱是在 0.0、0.5、1.0 和 2.0 存在的情况下获得的
Interleukin-2 (IL-2) is a 15.5 kDa cytokine that has a predominant role in the growth of activated T cells. IL-2 stimulates T-cell proliferation by binding on the T-cell surface with picomolar affinity to a heterotrimeric receptor complex consisting of R,, and γ chains. 1 Antibodies that recognize the R receptor subunit (IL-2RR) and block IL-2 binding have proven clinically effective as immunosuppressive agents, 1, 2 and thus, we have sought small molecules capable of blocking the IL-2/IL-2RR interaction as potential orally active successors to the antibody drugs. The design of such agents is based on a combination of structural information obtained by X-ray crystallographic3, 4 and NMR studies5 of IL-2 which reveal the backbone to include a bundle of four R-helixes (A, B, C, and D) with an up-up-down-down arrangement and site-directed mutagenesis of human IL-2 which identified residues in the AB loop (K35, R38, T41, F42, K43, Y45) and the B helix (E62, L72) as being critical for the binding of IL-2 to IL-2RR. 6, 7 One member of a series of acylphenylalanine derivatives intended to mimic the R38-F42 region of IL-2 was found to be a competitive inhibitor of IL-2/IL-2RR binding with a midmicromolar IC50. Structure-activity studies led to the synthesis of 1 with an IC50 of 3 µM. Although the acylphenylalanine derivatives were designed to complex with IL-2RR by emulating residues R38 and F42 of the IL-2 ligand, we considered the possibility that they might instead interact with the ligand. NMR studies using uniformly 15N-labeled IL-2 indicate that the resonances of residues in the vicinity of the binding epitope previously identified by site-directed mutagenesis are selectively perturbed in a concentration-dependent manner by 1, but not by its inactive enantiomer 2, indicating that 1 inhibits IL-2/IL-2RR binding by associating with IL-2. To our knowledge, this represents the first well-characterized example of a small molecule, nonpeptide inhibitor of a cytokine/cytokine receptor interaction.Compounds were evaluated for the ability to inhibit IL-2/IL-2RR interactions using a scintilation proximity competitive binding assay. 8 The assay basically measures binding of 125I-labeled IL-2 to immobilized soluble IL-2RR in the presence of various concentrations of inhibitor compound. As determined from Hill plots of the inhibition curves, compound 1 inhibited binding of IL-2 to IL-2RR with an IC50 of 3 µM at pH 7.4. In contrast, it’s enantiomer, 2, gave only 14% inhibition of binding at the highest concentration tested (500 µM), while IL-2 had an IC50 of 13 nM. To characterize the inhibitory activity of 1, binding assays were carried out with varying concentrations of 125I-labeled IL-2 in the presence of varying concentrations of 1 or unlabeled IL-2. Scatchard analyses were performed and the Kd values for [125I] IL-2 binding at each inhibitor concentration were plotted versus inhibitor concentration in a Schild plot9 (Figure 1), which indicates that the observed inhibitory activity of 1 is consistent with a competitive reversible inhibition. To assess the mechanism of inhibition, NMR experiments were undertaken with uniformly 15N-enriched IL-2. 10 The 1H-15N NMR assignments were obtained by analysis of 3D 1H-1H-15N-TOCSY-and NOESY-HSQC spectra. 11 These resonance assignments were, in most cases, determined to be similar to those previously published. 12 Perturbations of the 1H-15N chemical shifts were measured in 2D 1H-15N-HSQC spectra13 which were acquired in the presence of 0.0, 0.5, 1.0, and 2.0