Predicting Drug Interactions with Human Equilibrative Nucleoside Transporters 1 and 2 Using Functional Knockout Cell...

Area of Science:

• Pharmacology and Drug Discovery
• Molecular Biology
• Reproductive Medicine

Background:

• Equilibrative nucleoside transporters (ENTs) 1 and 2 are crucial for nucleoside transport across the blood-testis barrier (BTB).
• Targeting these endogenous transport pathways could enhance drug delivery to the testes for treating reproductive tract diseases.

Purpose of the Study:

• To generate and characterize CRISPR/CRISPR-associated protein 9-generated HeLa cell lines with limited ENT1 or ENT2 expression.
• To develop Bayesian models for predicting drug interactions with ENT1 and ENT2.
• To identify compounds that can utilize ENT1 and ENT2 to circumvent the BTB.

Main Methods:

• CRISPR/CRISPR-associated protein 9 was used to create ENT1 and ENT2 functional knockouts in HeLa S3 cells.
• Uridine transport was quantified in the presence of the ENT inhibitor NBMPR to assess transporter function.
• Bayesian models were generated using Assay Central machine learning software and validated with ChEMBL data.

Main Results:

• Twenty-one out of 46 evaluated compounds inhibited uridine uptake via ENT1 and/or ENT2.
• Abacavir, nevirapine, ticagrelor, and uridine triacetate exhibited differential IC50 values for ENT1 versus ENT2.
• Clofarabine and cladribine were identified as ENT1 and ENT2 substrates, while nevirapine and lexibulin were non-transported inhibitors.

Conclusions:

• This study presents the first Bayesian models for predicting drug interactions with ENT1 and ENT2.
• The developed models demonstrate good internal validation performance.
• Identifying drug interactions with ENTs aids in predicting substrates that can bypass the BTB via Sertoli cell transepithelial transport.