Folic Acid–Peptide Conjugates Combine Selective Cancer Cell Internalization with Thymidylate Synthase Dimer Interface Targeting

Drug–target
interaction, cellular internalization, and target engagement
should be addressed to design a lead with high chances of success
in further optimization stages. Accordingly, we have designed conjugates
of folic acid with anticancer peptides able to bind human thymidylate
synthase (hTS) and enter cancer cells through folate receptor α
(FRα) highly expressed by several cancer cells. Mechanistic
analyses and molecular modeling simulations have shown that these
conjugates bind the hTS monomer–monomer interface with affinities
over 20 times larger than the enzyme active site. When tested on several
cancer cell models, these conjugates exhibited FRα selectivity
at nanomolar concentrations. A similar selectivity was observed when
the conjugates were delivered in synergistic or additive combinations
with anticancer agents. At variance with 5-fluorouracil and other
anticancer drugs that target the hTS catalytic pocket, these conjugates
do not induce overexpression of this protein and can thus help combating
drug resistance associated with high hTS levels.

为确保在后续优化阶段获得较高的研发成功率，需针对药物-靶点相互作用（drug–target interaction）、细胞内化过程与靶点结合效应开展系统性研究。据此，我们设计了叶酸（folic acid）与抗癌肽的偶联物，该类偶联物可结合人胸苷酸合酶（hTS），并通过多种癌细胞高表达的叶酸受体α（FRα）进入癌细胞。机制分析与分子建模模拟结果显示，此类偶联物结合hTS的单体-单体界面的亲和力，较该酶活性位点高出20倍以上。在多种癌细胞模型中开展测试时，此类偶联物在纳摩尔浓度下即表现出对FRα的选择性。当此类偶联物与抗癌剂以协同或相加组合方式给药时，同样观测到了类似的选择性。与靶向hTS催化口袋的5-氟尿嘧啶（5-fluorouracil）及其他抗癌药物不同，此类偶联物不会诱导该蛋白的过表达，因此可助力对抗由高hTS水平引发的耐药性。