A Two-Prong Approach for Targeting the mRNA Cap-Dependent Translation Initiation: Small Molecules and Hydrocarbon Stapled Peptides

drug discovery high throughput screening;protein-protein interaction assay development;chemical biology;cap-dependent translation initiation;hydrocarbon stapled peptides targeting eIF4E interactions;protein-protein interactions;Astronomy;Atmospheric;Oceanic and Space Sciences;Biological Chemistry;Chemical Engineering;Chemistry;Science;Chemical Biology

Song, JamesSun, Duxin ;

University of Michigan

关键词: drug discovery high throughput screening;    protein-protein interaction assay development;    chemical biology;    cap-dependent translation initiation;    hydrocarbon stapled peptides targeting eIF4E interactions;    protein-protein interactions;    Astronomy;    Atmospheric;    Oceanic and Space Sciences;    Biological Chemistry;    Chemical Engineering;    Chemistry;    Science;    Chemical Biology;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/145870/jmsong_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Cap-dependent protein translation (CdT) is dysregulated in many types of cancer and leads to overexpression of oncogenes promoting angiogenesis, evasion of apoptosis, and cell proliferation. The protein-protein interactions (PPIs) involving eIF4E, 4E-BP1, and eIF4G1 dynamically regulate the initiation of the CdT and are therapeutic targets of interest in treatment of breast, pancreatic, ovarian, and prostate cancers; successful inhibition of the CdT could also provide selectivity towards targeting the protein translation addicted cancers over the healthy cells. In order to discover potent inhibitors of the CdT initiation, full-length eIF4E protein interactions were targeted using a two-pronged approach: small molecule discovered via high-throughput screening and rationally designed hydrocarbon stapled peptides.To conduct a high-throughput screening campaign, the assay platform catalytic enzyme-linked click chemistry assay (cat-ELCCA) was expanded to screen against full-length PPIs to create PPI cat-ELCCA and implemented for the eIF4E–4E-BP1 interaction. PPI cat-ELCCA exhibited over 10-fold improvement in limits of detection and quantification over ELISA and was successfully miniaturized using automated liquid handlers with exceptional assay parameters (Z’ > 0.6, signal-to-background > 30). Using PPI cat-ELCCA, over 50,000 natural product extracts (NPE) and custom compounds were screened, of which 18 NPE fractions and 9 compounds exhibited dose-dependent inhibition with hill slope ranging between -0.7 to -2.0. All the custom compounds were identified with micromolar inhibitory potency and 6 of the 9 compounds had demonstrated direct binding interaction to target protein eIF4E. Further re-isolation and iterative screening are pending for the active NPE fractions, and compound structure disclosures of hit molecules are awaiting approval.As an alternative drug discovery approach, the α-helical structure adopted by the 4E-BPs upon binding to eIF4E was exploited to design 4E-BP1 mimetic hydrocarbon stapled peptides (HCS). The lead HCS peptide HCS 4E-BP1 exhibited 5-fold greater inhibitory potency and eIF4E direct binding affinity (4 nM and 4 nM, respectively) than the linear 4E-BP1, accompanied by a 250% increase in peptide helicity. HCS 4E-BP1 successfully inhibited eIF4E PPIs with both 4E-BP1 and eIF4G1 in a dose-dependent manner in cellulo in presence of serum. Overall, the results from this two-pronged eIF4E inhibitor discovery campaign have pushed forward the current limits of targeting the CdT initiation and produced promising leads for further probe and drug development.