TSPO ligand-methotrexate prodrug conjugates: Design, synthesis, and biological evaluation

Valentino Laquintana, Nunzio Denora, Annalisa Cutrignelli, Mara Perrone, Rosamaria Iacobazzi, Cosimo Annese, Antonio Lopalco, Angelaassunta Lopedota, Massimo Franco

Research output: Contribution to journalArticlepeer-review


The 18-kDa translocator protein (TSPO) is a potential mitochondrial target for drug delivery to tumors overexpressing TSPO, including brain cancers, and selective TSPO ligands have been successfully used to selectively deliver drugs into the target. Methotrexate (MTX) is an anticancer drug of choice for the treatment of several cancers, but its permeability through the blood brain barrier (BBB) is poor, making it unsuitable for the treatment of brain tumors. Therefore, in this study, MTX was selected to achieve two TSPO ligand-MTX conjugates (TSPO ligand α-MTX and TSPO ligand γMTX), potentially useful for the treatment of TSPO-rich cancers, including brain tumors. In this work, we have presented the synthesis, the physicochemical characterizations, as well as the in vitro stabilities of the new TSPO ligand-MTX conjugates. The binding affinity for TSPO and the selectivity versus central-type benzodiazepine receptor (CBR) was also investigated. The cytotoxicity of prepared conjugates was evaluated on MTX-sensitive human and rat glioma cell lines overexpressing TSPO. The estimated coefficients of lipophilicity and the stability studies of the conjugates confirm that the synthesized molecules are stable enough in buffer solution at pH 7.4, as well in physiological medium, and show an increased lipophilicity compared to the MTX, compatible with a likely ability to cross the blood brain barrier. The latter feature of two TSPO ligand-MTX conjugates was also confirmed by in vitro permeability studies conducted on Madin-Darby canine kidney cells transfected with the human MDR1 gene (MDCK-MDR1) monolayers. TSPO ligand-MTX conjugates have shown to possess a high binding affinity for TSPO, with IC50 values ranging from 7.2 to 40.3 nM, and exhibited marked toxicity against glioma cells overexpressing TSPO, in comparison with the parent drug MTX.

Original languageEnglish
Article number967
JournalInternational Journal of Molecular Sciences
Issue number6
Publication statusPublished - Jun 1 2016


  • Bio-conjugate
  • Glioma
  • Methotrexate
  • Translocator protein
  • TSPO-ligand

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Spectroscopy
  • Inorganic Chemistry
  • Catalysis
  • Molecular Biology
  • Computer Science Applications


Dive into the research topics of 'TSPO ligand-methotrexate prodrug conjugates: Design, synthesis, and biological evaluation'. Together they form a unique fingerprint.

Cite this