MATE2-K Inhibition Assessment Service

Precision MATE2 Inhibition Assessment

Drug transporters are integral components of cellular membranes responsible for facilitating the movement of various substances across the cell. Multidrug and toxin extrusion protein 2-K (MATE2-K) is a member of the solute carrier (SLC) superfamily and plays a crucial role in the active renal secretion of drugs.

At Creative Biolabs, in our cell-based assay, we aim to assess whether test compounds act as inhibitors of specific target transporters, with a particular focus on MATE2-K. To evaluate inhibition potential, we employ metformin, a well-established substrate of organic anion transporter 1 (OAT1), as the probe substrate. Metformin's intracellular accumulation in a cell line overexpressing MATE2-K is detected using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). This method allows us to quantify the level of metformin accumulation, serving as a proxy for transporter activity.

Detailed Methods

Cell Culture and Treatment	Use a cell line CHO-K1/HEK293 that overexpresses MATE2-K. These cells are genetically engineered to stably overexpress MATE2-K, ensuring that the observed effects are due to this specific transporter.
Reagents and Preparation	Probe Substrate: metformin Test Compounds: Select the compounds to be tested, dissolve them in an appropriate solvent, and dilute them to the desired working concentration.
Experimental Procedure	1. Cell seeding 2. Pre-incubation 3. Compound treatment 4. Probe substrate treatment 5. Termination and washing
Sample Preparation	Cell lysis and sample collection
LC-MS/MS Analysis	Standard Curve: Prepare a series of metformin standard solutions with known concentrations for quantification. Sample Analysis: Quantify the intracellular metformin using LC-MS/MS. Data Analysis: Compare the metformin concentrations between different treatment groups to evaluate the inhibitory effect of the test compounds on MATE2-K.
Data Processing	Calculate the metformin concentration for each sample(n=2).

The principle underlying this assay is based on the inverse relationship between the level of test compound transporter inhibition activity and the accumulation of the probe substrate. When the test compound blocks MATE2-K, intracellular accumulation of metformin is reduced. Testing the compound on MATE2-K activity at 8 concentrations replicated twice (n=2) will determine the half-maximal inhibitory concentration (IC50) of this compound in inhibiting MATE2-K function.

In summary, the development of this assay helps to assess test compounds for their potential to inhibit MATE2-K-mediated drug transport. Our findings make an important contribution toward characterizing inhibitor effects in a key transporter and support efforts to predict and prevent drug-drug interactions as well as design safer, more efficacious therapeutic agents.

DAPI (4',6-diamidino-2-phenylindole) also serves as a fluorescent substrate that can be transported by MATE2-K. By monitoring the uptake and efflux of DAPI in cells expressing MATE2-K, researchers can evaluate the inhibitory effects of various compounds on MATE2-K activity.

Assay Principle:

Cells expressing MATE2-K are incubated with DAPI and potential inhibitors.
DAPI uptake and efflux are measured to determine the transport activity of MATE2-K.
In the presence of an effective MATE2-K inhibitor, the transport of DAPI is reduced, leading to increased intracellular DAPI fluorescence.

Applications

Drug Development: Understanding the interaction between new drug candidates and MATE2 transporters.
Safety Assessment: Evaluating potential drug-drug interactions and toxicities.
Personalized Medicine: Informing dose adjustments and treatment plans based on transporter activity.

For Research Use Only | Not For Clinical Use