Inhibition of sodium/hydrogen exchanger 3 in the gastrointestinal tract by tenapanor reduces paracellular phosphate permeability.

Hyperphosphatemia is common in patients with chronic kidney disease and is increasingly associated with poor clinical outcomes. Current management of hyperphosphatemia with dietary restriction and oral phosphate binders often proves inadequate. Tenapanor, a minimally absorbed, small-molecule inhibitor of the sodium/hydrogen exchanger isoform 3 (NHE3), acts locally in the gastrointestinal tract to inhibit sodium absorption. Because tenapanor also reduces intestinal phosphate absorption, it may have potential as a therapy for hyperphosphatemia. We investigated the mechanism by which tenapanor reduces gastrointestinal phosphate uptake, using in vivo studies in rodents and translational experiments on human small intestinal stem cell-derived enteroid monolayers to model ion transport physiology. We found that tenapanor produces its effect by modulating tight junctions, which increases transepithelial electrical resistance (TEER) and reduces permeability to phosphate, reducing paracellular phosphate absorption. NHE3-deficient monolayers mimicked the phosphate phenotype of tenapanor treatment, and tenapanor did not affect TEER or phosphate flux in the absence of NHE3. Tenapanor also prevents active transcellular phosphate absorption compensation by decreasing the expression of NaPi2b, the major active intestinal phosphate transporter. In healthy human volunteers, tenapanor (15 mg, given twice daily for 4 days) increased stool phosphorus and decreased urinary phosphorus excretion. We determined that tenapanor reduces intestinal phosphate absorption predominantly through reduction of passive paracellular phosphate flux, an effect mediated exclusively via on-target NHE3 inhibition.

Characterization of G-protein coupled receptor modulators using homogeneous cAMP assays.

More than two-thirds of all known G-protein coupled receptors are known to modulate the function of adenylate cyclase resulting in altered levels of cAMP. In turn, cAMP fluctuations transform agonist binding events into physiological changes in cell behavior. The advent of nonradioactive, homogeneous methods of measuring intracellular cAMP has enabled the rapid growth of drug discovery and research applications for these GPCR targets. In this chapter, we describe a nonradioactive, chemiluminescent cAMP detection method using enzyme fragment complementation technology to detect a wide range of GPCR modulators which is also suitable for high-throughput screening.

Importance of calcium-binding site 2 in simian virus 40 infection.

The exposure of molecular signals for simian virus 40 (SV40) cell entry and nuclear entry has been postulated to involve calcium coordination at two sites on the capsid made of Vp1. The role of calcium-binding site 2 in SV40 infection was examined by analyzing four single mutants of site 2, the Glu160Lys, Glu160Arg, Glu157Lys (E157K), and Glu157Arg mutants, and an E157K-E330K combination mutant. The last three mutants were nonviable. All mutants replicated viral DNA normally, and all except the last two produced particles containing all three capsid proteins and viral DNA. The defect of the site 1-site 2 E157K-E330K double mutant implies that at least one of the sites is required for particle assembly in vivo. The nonviable E157K particles, about 10% larger in diameter than the wild type, were able to enter cells but did not lead to T-antigen expression. Cell-internalized E157K DNA effectively coimmunoprecipitated with anti-Vp1 antibody, but little of the DNA did so with anti-Vp3 antibody, and none was detected in anti-importin immunoprecipitate. Yet, a substantial amount of Vp3 was present in anti-Vp1 immune complexes, suggesting that internalized E157K particles are ineffective at exposing Vp3. Our data show that E157K mutant infection is blocked at a stage prior to the interaction of the Vp3 nuclear localization signal with importins, consistent with a role for calcium-binding site 2 in postentry steps leading to the nuclear import of the infecting SV40.

Molecular dissection of nuclear entry-competent SV40 during infection.

To establish viral infection, SV40 must expose nuclear localization signals (NLSs) that are internal in the virion architecture in order to enter the nucleus via interaction with the host's nuclear import machinery, which includes importin alpha and importin beta. The time course for SV40 association with the importins in infected cells was examined. The viral DNA associated with importin alpha by 1.5h post infection, before associating with the importin beta nuclear import receptor, by 3h post infection. Only a small fraction of cell-internalized SV40 that contained viral DNA was bound by the two importins. This fraction, termed "nuclear entry-competent SV40," was slightly smaller than the virion but, importantly, was larger than the viral chromatin and contained both Vp1 and Vp3. Furthermore, the internalized viral DNA in either anti-importin or anti-Vp3 immune complexes was sensitive to DNase I, whereas the viral DNA in mature virions was resistant. All these results suggest that once SV40 enters the cytoplasm, it undergoes an architectural modification that exposes the virion's NLSs for nuclear entry.