Interaction of the Novel Adenosine Uptake Inhibitor 3-[1-(6,7-Diethoxy-2-morpholinoquinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1H,3H)-quinazolinedione Hydrochloride (KF24345) with the es and ei Subtypes of Equilibrative Nucleoside Transporters

摘要

Nucleosides such as adenosine,as well as many nucleoside-based drugs,permeate cell membranes via a family of equilibrative nucle-oside transporters (ENTs).We assessed the effects of (3-[1-(6,7-diethoxy-2-morpholino-quinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1H,3H)-quinazolinedione hydrochloride (KF24345),a novel anti-inflammatory agent that potentiates the actions of adenosine,on the es (inhibitor-sensitive) and ei (inhibitor-resistant) subtypes of ENTs in human,mouse,and rat cells.KF24345 was similar to the prototypical high-affinity inhibitor nitrobenzylthioinosine (NBMPR) for blocking the human es transporter (K,of approx 0.4 nM),but was 50-fold more effective than NBMPR at blocking the human ei transporter (K_I of -100 nM).KF24345 displayed significantly less species heteroge-neity in its affinity for the es transporter than did dipyridamole,a widely used inhibitor of nucleoside transport;KF24345 may thus prove useful as an inhibitor for studies of nucleoside metabolism in a range of animal models.Furthermore,KF24345 seemed to act as a noncompetitive inhibitor of both pHJNBMPR binding and pH]nucleo-side uptake by human es transporters,and these kinetics were con-sistent with an observed slow dissociation of KF24345 from the inhibitor binding site.KF24345 also exhibited unusual biphasic pro-files for inhibition of pH]NBMPR binding to membranes prepared from a recombinant human es transporter model (PK15-hENT1),suggesting the presence of multiple populations of NBMPR binding proteins in these membranes.The atypical tight binding interaction of KF24345 with the es transporter may prove useful for the molecular delineation of inhibitor binding domains and will facilitate its use as an in vivo inhibitor of nucleoside transport in studies focused on the biological effects of adenosine.