G-quadruplexes are promising targets for innovative anticancer therapy. Hence, many efforts are being made to find selective ligands. Drug design is often based on the available high-resolution structures, obtained for the thermodynamically stable forms. However, the complexity of the G-quadruplex folding landscape has clearly emerged in recent years, with the discovery of intermediate conformations that persist on the second to the minute time scale. In the case of the KIT2 G-quadruplex forming sequence, found within human c-KIT promoter, we recently identified a long-lived folding intermediate, characterized by guanine stacking in alternating orientation (as determined by circular dichroism). Given the rate of transcriptional processes, a physiological role of this arrangement should not be excluded. In the present study, we applied circular dichroism (CD) spectroscopy, native electrospray ionization mass spectrometry (ESI-MS) and electrophoretic mobility shift assays (EMSA) to show that a perylene derivative (K20) selects this topology. Interestingly, ESI-MS spectra revealed the presence of a single specifically coordinated K⁺ ion in the structure, which is thus presumably composed of only two consecutive G-quartets. The parent ligand PIPER failed to promote the same conformational selection, which is therefore a process strictly dependent on the perylene side chains composition. The greater affinity of K20 for the two-quartet antiparallel topology, compared to PIPER, was finally corroborated by evaluating their binding to the KIT∗ G-quadruplex, which is also found within the human promoter of c-KIT.

G-四链体是创新抗癌治疗的有希望的目标。因此，为寻找选择性配体付出了许多努力。药物设计通常基于为热力学稳定形式获得的高分辨率结构。然而，近年来，随着四分之一秒尺度上持续存在的中间构象的发现，G四联体折叠景观的复杂性已经明显地出现。在人类c-KIT内发现的KIT2 G四联体形成序列的情况下启动子，我们最近确定了一个长寿的折叠中间体，其特征在于鸟嘌呤以交替方向堆叠（由圆二色性确定）。给定转录过程的速率，不应排除这种安排的生理作用。在本研究中，我们应用了圆二色性（CD）光谱，天然电喷雾电离质谱（ESI-MS）和电泳迁移率漂移测定（EMSA）来表明a衍生物（K20）选择了这种拓扑。有趣的是，ESI-MS光谱显示存在一个特定的专门配位的K ⁺结构中的离子，因此大概只由两个连续的G四重唱组成。母体配体PIPER无法促进相同的构象选择，因此，该过程严格取决于the侧链的组成。通过评估它们与KIT * G-四链体的结合，最终证实了与PIPER相比，K20对两重四体反平行拓扑结构具有更大的亲和力，该亲和力也在c-KIT的人类启动子中发现。