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KW-7158 is a novel therapeutic candidate for treating overactive bladder (OAB) with a unique mode of action: suppression of sensory afferent nerves. However, the molecular target of this compound remains unknown. We herein report the identification of the KW-7158 target to be equilibrative nucleoside transporter-1 (ENT1). A membrane protein expression library of ca. 7000 genes was expressed in a dorsal root ganglion cell line, which we had previously generated, and subjected to screening for binding with a fluorescent derivative that retains high binding activity to the target. The screening revealed that only cells transfected with an ENT1 expression vector exhibited significant binding. We next performed [ 3 H]KW-7158 binding experiments and an adenosine influx assay and found that KW-7158 binds to and inhibits ENT1. To further demonstrate the pharmacological relevance, we evaluated other known ENT1 inhibitors (nitrobenzylthioinosine, dipyridamole, draflazine) in an in vitro bladder strip contraction assay and the rat spinal cord injury OAB model. We found that all of the inhibitors exhibited anti-OAB activities, of which the potencies were comparable to that of adenosine influx inhibition in vitro. These studies demonstrated that the pharmacological target of KW-7158 is ENT1, at least in the rat OAB model. Our results will aid understanding of the precise mechanism of action of this drug and may also shed new light on the use of the adenosine pathway for the treatment of OAB.Key words nociceptive neuron; overactive bladder; adenosine transporter; drug target identification KW-7158 is a drug candidate for overactive bladder (OAB).1-3) Pharmacological studies using rats with xyleneirritated bladders, 4) cultured dorsal root ganglion (DRG) neurons, 5) and isolated bladder strips 6) suggest that KW-7158 has a unique mechanism of action, namely, it inhibits the contraction of the bladder smooth muscle by suppressing the activity of sensory nerves. This unique mode of action distinguishes KW-7158 as a drug candidate that may be free from the side effects caused by other anti-OAB drugs. However, the molecular target of KW-7158 remains elusive.We have previously shown that the target exists in the rat DRG using [ 3 H] KW-7158 radioligand binding assays. 6) To facilitate target identification, we have generated various DRG neuron-like cell lines, including TRD-10 cells, which express a large amount of the target, and TRD-49 cells, which express a negligible amount of the target. 6)In this study, using the DRG cell lines and a KW-7158 fluorescent derivative, we first screened the membrane protein expression library to identify clones expressing KW-7158 binding molecules. This resulted in the identification of a single clone that codes equilibrative nucleoside transporter-1 (ENT1). Further in vitro and in vivo pharmacological studies demonstrated that KW-7158 is a novel ENT1 inhibitor and that the inhibition of ENT1 counteracts overactive bladder in the rat spinal cord injury OAB model. MATERIALS AND METHODSReagents KW-...