Virtual Screening and Optimization of Novel mTOR Inhibitors for Radiosensitization of Hepatocellular Carcinoma

Abstract

Background

Radiotherapy has an ameliorative effect on a wide variety of tumors, but hepatocellular carcinoma (HCC) is insensitive to this treatment. Overactivated mammalian target of rapamycin (mTOR) plays an important part in the resistance of HCC to radiotherapy; thus, mTOR inhibitors have potential as novel radiosensitizers to enhance the efficacy of radiotherapy for HCC.

Methods

A lead compound was found based on pharmacophore modeling and molecular docking, and optimized according to the differences between the ATP-binding pockets of mTOR and PI3K. The radiosensitizing effect of the optimized compound (2a) was confirmed by colony formation assays and DNA double-strand break assays in vitro. The discovery and preclinical characteristics of this compound are described.

Results

The key amino acid residues in mTOR were identified, and a precise virtual screening model was constructed. Compound 2a, with a 4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine scaffold, exhibited promising potency against mTOR (mTOR IC₅₀=7.1 nmol/L (nM)) with 126-fold selectivity over PI3Kα. Moreover, 2a significantly enhanced the sensitivity of HCC to radiotherapy in vitro in a dose-dependent manner.

Conclusion

A new class of selective mTOR inhibitors was developed and their radiosensitization effects were confirmed. This study also provides a basis for developing mTOR-specific inhibitors for use as radiosensitizers for HCC radiotherapy.

Keywords:

• virtual docking
• HCC
• kinase inhibitor
• mTOR
• radiosensitizer

Supplementary Materials

Figure S1–S3; MD simulations (Figure S4–S6); Western blot assays (Figure S7); Figure S8; the ¹H-NMR, ¹³C-NMR and MS spectra of compound 1a–1d, 2a–2j and 3a–3h.

Acknowledgments

We gratefully acknowledge financial support from the program for the National Natural Science Foundation of China-Youth Foundation (No. 81502906), Graduate Innovative Fund of Wuhan Institute of Technology (No. CX2018001) and Natural Science Foundation of Beijing, China (No. 7172205).

Abbreviations

HCC, hepatocellular carcinoma; mTOR, mammalian target of rapamycin; ATP, adenosine triphosphate; PI3K, phosphoinositide 3-kinase; nM, nmol/L; μM, μmol/L; mM, mmol/L; PIKK, phosphoinositide 3-kinase-related kinase; DNA, deoxyribonucleic acid; ADP, adenosine diphosphate; TR-FRET, time-resolved fluorescence resonance energy transfer; IR, ionizing radiation; EDTA, ethylene diamine tetraacetic acid; DMSO, dimethyl sulfoxide; 4EBP1, 4E-binding protein 1; RLU, relative light unit; DMEM, Dulbecco’s modified eagle medium; O.D., optical density; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; MD, molecular dynamics; DSBs, double-strand breaks.

Author Contributions

All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.