| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2002: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2001: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
To apply a nutrient transporter for the drug delivery, we characterized an oligopeptide transporter (PEPT1) as well as a novel monocarboxylate transporter. The rationale behind this delivery approach is that these transporters show a broad substrate specificity and they can take up structurally modified drugs.
At first, we demonstrated using dipeptide analogues conjugated to linear aliphatic acids with various length that the affinity of analogues for PEPT1 is more potent proportional to the length of aliphatic acids. This indicated that the hydrophobicity is one of factors in the substrate recognition by PEPT1. To ensure the hydrophobic region around a recognition site, we synthesized dipeptide analogues conjugating ?A-amino group of Lys in Val-Lys with an environment-sensitive fluorescent probe, 2-anilino, 6-naphthalene sulfonate (ANS), which increase a fluorescent intensity reflecting a hydrophobic environment. We demonstrated that the analogue appreciably enhanced the fluorescence i
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n the presence of rat BBMVs or Caco-2 cells, indicating the hydrophobic region certainly locating in or near the recognition in PEPT1.
Secondly, we established stably transfected CHO cells over-exoressine the human H^+/dipeptide contransporter PEPT1 (designated CHO/hPEPT1). The accumulation of a PEPT1 substrate Gly-Sar by CHO/hPEPTl1 cells showed more than one hundred higher than that by the control CHO cells. This enabled us to analyze the molecular machinery of hPEPT1 in detail. Electrophysiological analysis showed a transient current coupling to a transport of Gly-Sar. Further, it was demonstrated that the uptake of substrate is voltage-dependent, whereas the efflux is independent. The CHO/hPEPT1 system would be a useful tool to screen whether hPEPT1 can take up the structurally modified drug as well as to investigate the detail mechanism of transport cycle (influx and efflux).
In addition, we started a research for the elucidation of molecular mechanism of multispecific transporter, EmrE, a small multidrug transporter in Escherichia coli. By using a site-directed mutagenesis and a chemical modification, we demonstrated that Thr56 locating in a center of the transporter is responsible for the substrate selectivity, and that the loop2-3 including the residue alters the conformation in a process of ethidium extrusion. These findings would be a good help to clarify the multispecific transporter, leading us to a fine development of an expectative DDS. Less
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