In vitro activity: PPQ-102 (also known as CFTR Inhibitor) is a potent inhibitor of CFTR (cystic fibrosis transmembrane conductance regulator). It can slow cyst enlargement in polycystic kidney disease and reduce intestinal fluid loss in secretory diarrheas. PPQ-102 completely inhibited CFTR chloride current with an IC50 of approximately 90 nM. Unlike prior CFTR inhibitors, PPQ-102 is uncharged at physiological pH, and therefore not subject to membrane potential-dependent cellular partitioning or block efficiency. Patch-clamp analysis confirmed voltage-independent CFTR inhibition by PPQ-102 and showed stabilization of the channel closed state. PPQ-102 prevented cyst expansion and reduced the size of preformed cysts in a neonatal kidney organ culture model of polycystic kidney disease. PPQ-102 is the most potent CFTR inhibitor identified to date.

Patch-Clamp Analysis: Whole cell recordings were done on FRT cells stably expressing human wild-type CFTR. The pipet solution contained 140 mM N-methyl d-glucamine chloride (NMDG-Cl), 5 mM EGTA, 1 mM MgCl2, 1 mM Tris-ATP, and 10 mM HEPES (pH 7.2), and the bath solution contained 140 mM N-methyl d-glucamine chloride, 1 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4). Experiments were done at room temperature (22–25 °C). Pipettes were pulled from borosilicate glass and had resistances of 3–5 MΩ after fire polishing. Seal resistances were typically between 3 and 10 GΩ. After establishing the whole-cell configuration, CFTR was activated by adding forskolin and 3-isobutyl-1-methylxanthine (IBMX). Whole cell currents were elicited by applying hyperpolarizing and depolarizing voltage pulses from a holding potential of 0 mV to potentials between –100 and +100 mV in steps of 20 mV. Current was filtered at 5 kHz and digitized and analyzed using an AxoScope 10.0 system and a Digidata 1440A AC/DC converter. The single channel characteristics of CFTR were analyzed in the cell-attached configuration using fire-polished pipettes with a resistance of 10–15 MΩ. The pipet solution contained (in mM): 140 NMDG-Cl, 1 CaCl2, 1 MgCl2, 5 glucose, and 10 HEPES (pH 7.4), and the bath solution contained 140 KCl, 1 CaCl2, 1 MgCl2, 5 glucose, and 10 HEPES (pH 7.4). Recordings were performed at room temperature using an Axopatch-200B (Axon Instruments). The voltage and current data were low-pass filtered at 1 kHz and stored for later analysis. Single channel data were digitally filtered at 25 Hz and analyzed using Clampfit 10.0 software.

Cell Assay: Airway epithelial NCI-H292 cells and primary cultures of noncystic fibrosis human airway epithelial cells were treated with cystic fibrosis transmembrane conductance regulator (CFTR) inhibitors (CFTR-inh(172) or PPQ-102) or transfected with a CFTR small interfering (si)RNA with or without a selective epidermal growth factor receptor tyrosine kinase inhibitor [3]. in vivo: PPQ-102 prevented cyst expansion and reduced the size of preformed cysts in a neonatal kidney organ culture model of polycystic kidney disease.