A novel high-throughput pepT1 transporter assay differentiates between substrates and antagonists.

Teresa N. Faria, Julita K. Timoszyk, Terry R. Stouch, Balvinder S. Vig, Christopher P. Landowski, Gordon L. Amidon, C. David Weaver, Doris A. Wall, Ronald L. Smith

Research output: Contribution to journalArticleScientificpeer-review

39 Citations (Scopus)


PepT1 is a transporter of proven pharmaceutical utility for enhancing oral absorption. A high-throughput, robust functional assay, capable of distinguishing PepT1 binders from substrates, allowing identification and/or prediction of drug candidate activation was developed. An MDCK epithelial cell line was transfected with rPepT1. The high level of stable rPepT1 expression that was achieved enabled development of a miniaturized PepT1 assay in a 96-well format, which could be scaled to 384 wells. The assay is based on measurement of membrane depolarization resulting from the cotransport of protons and PepT1 substrates. Membrane potential changes are tracked with a voltage-sensitive fluorescent indicator. Control (mock-transfected) cells are used to determine nonspecific membrane potential changes. A variety of fluorescent dyes were tested during initial assay design, including intracellular pH and membrane potential indicators. A membrane potential indicator was chosen because of its superior performance. Upon PepT1 activation with glycylsarcosine, dose-dependent membrane depolarization was observed with an EC50 of 0.49 mM. Maximum depolarization was dependent on the level of PepT1 expression. Testing of 38 known PepT1 substrates, binders, and nonbinders demonstrated that this assay accurately distinguished substrates from binders and from nonbinders. Initial validation of this novel assay indicates that it is sensitive and robust, and can distinguish between transporter substrates and antagonists. This important distinction has been previously achieved only with lower-throughput assays. This assay might also be used to determine substrate potency and establish a high-quality data set for PepT1 SAR modeling.

Original languageEnglish
Pages (from-to)67-76
Number of pages10
JournalMolecular Pharmaceutics
Issue number1
Publication statusPublished - 1 Jan 2004
MoE publication typeA1 Journal article-refereed


Dive into the research topics of 'A novel high-throughput pepT1 transporter assay differentiates between substrates and antagonists.'. Together they form a unique fingerprint.

Cite this