Details

Autor(en) / Beteiligte

• Zachos, Nicholas C.
• vaughan, hannah
• Sarker, Rafiquel
• Lin, Ruxian
• Tse, C Ming
• Green, Jordan J.
• Donowitz, Mark

Titel

266 NHE3 IS A DRUG TARGET FOR TREATING DIARRHE; A NOVEL PEPTIDE STIMULATES NHE3 ACTIVITY, PREVENTS CAMP, CGMP AND CA2+ INHIBITION OF NHE3 AND PREVENTS CHOLERA TOXIN AND E. COLI STABLE ENTEROTOXIN FLUID SECRETION

Ist Teil von

• Gastroenterology (New York, N.Y. 1943), 2021-05, Vol.160 (6), p.S-63-S-64

Ort / Verlag

Elsevier Inc

Erscheinungsjahr

2021

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• While diarrheal diseases remain a world-wide cause of mortality and morbidity, pharmacologic therapy remains ineffectual for severe cases. Reduced intestinal Na absorption is part of the pathophysiology of diarrheal diseases and is due to inhibition of the BB Na/H exchanger NHE3 in nearly all diarrhea diseases. NHE3 is highly regulated, which involves large multi-protein complexes on its intracellular domain, and includes an inhibitory complex. We tested the hypothesis that a peptide that competes with the NHE3 inhibitory complex would stimulate NHE3 activity and could represent a new drug to treat diarrhea. Peptides mimicking the NHE3 inhibitory domain (38aa called N3SP-1) and several shorter peptides were synthesized and loaded into intestinal cells using Bodipy 57/618 (maleimide) or bioreducible carboxylated poly (beta-amino ester) polymers (cPBAE) that complexed with the peptides to form self-assembled nanoparticles. The latter was used to develop a system that could be used for intracellular peptide delivery in patients. NHE3 activity under basal and cAMP, cGMP, elevated Ca2+ conditions was measured in Caco-2 cells, mouse jejunum, human jejunal enteroids using BCECF or SNARF-4F fluorometry. Mouse small intestinal fluid secretion was determined using a perfused system and with weight based volume determination. cAMP was determined by ELISA. 1) Two control peptide made to the NHE3 inhibitory domain did not alter basal NHE3 activity and were used as negative controls. 2) N3SP-1 conjugated to Bodipy 57/618 loaded (15min, 37C) into Caco-2 cells, mouse jejunum in vitro, and human enteroids as visualized by fluorescence stimulated basal NHE3 activity by 40-60% (Ic50 150nM in Caco-2 cells). N3SP-1 reversed the forskolin and carbachol inhibition of NHE3 but did not alter NHE3 stimulation by EGF. 3) In vivo perfusion of mouse jejunum with N3SP-1 stimulated NHE3 activity and incubation of N3SP-1 with cholera toxin or E. coli heat stable enterotoxin prevented toxin stimulated fluid stimulation. 5) cPBAE-peptide nanoparticles were loaded into Caco-2 and human jejunal enteroids studied after overnight exposure. N3SP-1 (250 nM) stimulated NHE3 activity by 90% in Caco-2 cells and 40% in human jejunal enteroids. N3SP-1 also reversed the forskolin inhibition of NHE3 by 90% in human enteroids but did not alter the forskolin stimulated of cAMP. 1) A 38aa peptide mimicking the NHE3 inhibitory domain stimulates NHE3 activity in multiple cells types, both in vivo and in vitro, with a low Ic50 (150 nM), reverses the inhibition of NHE3 by elevated cAMP, cGMP and Ca2+ without altering second messenger effects. 2) Loading into intestinal cells with bioreducible polymer nanoparticles with similar transport effects shows that NHE3 is a druggable target for diarrheal diseases with potential for use in patients.