G‑Quadruplex Identification in the Genome of Protozoan Parasites Points to Naphthalene Diimide Ligands as New Antiparasitic Agents

G-quadruplexes (G4) are DNA secondary structures that take part in the regulation of gene expression. Putative G4 forming sequences (PQS) have been reported in mammals, yeast, bacteria, and viruses. Here, we present PQS searches on the genomes of T. brucei, L. major, and P. falciparum. We found telomeric sequences and new PQS motifs. Biophysical experiments showed that EBR1, a 29 nucleotide long highly repeated PQS in T. brucei, forms a stable G4 structure. G4 ligands based on carbohydrate conjugated naphthalene diimides (carb-NDIs) that bind G4’s including hTel could bind EBR1 with selectivity versus dsDNA. These ligands showed important antiparasitic activity. IC50 values were in the nanomolar range against T. brucei with high selectivity against MRC-5 human cells. Confocal microscopy confirmed these ligands localize in the nucleus and kinetoplast of T. brucei suggesting they can reach their potential G4 targets. Cytotoxicity and zebrafish toxicity studies revealed sugar conjugation reduces intrinsic toxicity of NDIs.G-四链体（G-quadruplexes，G4）是参与基因表达调控的DNA二级结构。现有研究已在哺乳动物、酵母、细菌及病毒中报道了潜在G4形成序列（Putative G4 forming sequences，PQS）。本研究针对布氏锥虫（Trypanosoma brucei，T. brucei）、利什曼原虫（Leishmania major，L. major）与恶性疟原虫（Plasmodium falciparum，P. falciparum）的基因组开展了PQS搜索，鉴定得到端粒序列与新型PQS基序。生物物理实验证实，布氏锥虫中一段长29个核苷酸的高度重复PQS序列EBR1可形成稳定的G4结构。基于碳水化合物缀合萘二酰亚胺（carbohydrate conjugated naphthalene diimides，carb-NDIs）的G4配体，能够结合包括人端粒（hTel）在内的G4结构，且相较于双链DNA（dsDNA），对EBR1具有结合选择性。此类配体展现出显著的抗寄生虫活性，针对布氏锥虫的半最大抑制浓度（half maximal inhibitory concentration，IC50）处于纳摩尔级别，且对人MRC-5细胞具有高选择性。共聚焦显微镜（confocal microscopy）成像证实，此类配体可定位于布氏锥虫的细胞核与动基体（kinetoplast），提示其可抵达潜在的G4靶标位点。细胞毒性与斑马鱼毒性研究表明，碳水化合物缀合修饰可降低萘二酰亚胺类化合物的固有毒性。