Competitive Counterflow (CCF) To Distinguish Between Transported And Non‐Transported Inhibitors Of MATE1 and MATE2‐K

Abstract

The MATEs (hMATE1 & hMATE‐2K) are membrane proteins expressed in the apical membrane of renal proximal tubule cells that mediate the flux of organic cations into the filtrate. In this study we set to test CCF as a method to identify substrates of pharmacological interest for both MATEs.We first determined IC50 values for inhibition of MPP transport into CHO cells that stably expressed either MATE1 or MATE2‐K for several clinically important compounds: Amlodipine (IC50= 7‐6 μM), Atenolol (10‐4 μM), Dofetilidae (4‐0.8 μM), Entecavir (302‐140 μM), Lamivudine (336‐53 μM), Omeprazole (53‐8 μM), Propranolol, (16‐5 μM) and Trimethoprim (6‐0.4 μM). For CCF, cells were exposed to ~12 nM [3H]MPP for 40 min, sufficient to reach steady state. At time 0, the medium was removed and replaced with medium containing either ~12 nM [3H]MPP (control) or ~12 nM [3H]MPP plus one of the test compounds, or a known MATE substrate (e.g., Metformin); PAH (1mM) was used as a known non‐transported compound. After an additional 5 min incubation, radiolabeled MPP remaining in the cells was determined. If the extracellular test compound was transported, the associated turnover of the transporter should result in net loss of labeled MPP from the cells. Each test was performed (in sextuplicate) in 2–3 separate experiments.Whereas exposure to 1mM PAH resulted in no loss of MPP from the cells (which was distributed to steady state), exposure to metformin resulted in net loss of radiolabeled substrate (70%). Of the test compounds, Atenolol, Amlodipine & Lamivudine supported a net loss of [3H]MPP (70‐30%) from both MATE1 & MATE2K‐expressing cells, whereas Entecavir reduced total accumulation of MPP only for MATE2K expressing cells. There was no loss (or gain) of MPP induced by Dofetilidae, Propranolol or Omeprazole, suggesting they are non‐transported inhibitors for both MATEs.These data support the conclusion that the method of CCF is applicable for identification of MATE substrates, and identified a difference in the selectivity profile of hMATE1 & hMATE2K.Support or Funding InformationSupported by NIH Award 5R01GM129777