The evolutionary divergence of receptor guanylyl cyclase C has implications for preclinical models for receptor-directed therapeutics.

Mutations in receptor guanylyl cyclase C (GC-C) cause severe gastrointestinal disease, including meconium ileus, early onset acute diarrhea, and pediatric inflammatory bowel disease that continues into adulthood. Agonists of GC-C are US Food and Drug Administration-approved drugs for the treatment of constipation and irritable bowel syndrome. Therapeutic strategies targeting GC-C are tested in preclinical mouse models, assuming that murine GC-C mimics human GC-C in its biochemical properties and downstream signaling events. Here, we reveal important differences in ligand-binding affinity and GC activity between mouse GC-C and human GC-C. We generated a series of chimeric constructs of various domains of human and mouse GC-C to show that the extracellular domain of mouse GC-C contributed to log-orders lower affinity of mouse GC-C for ligands than human GC-C. Further, the Vmax of the murine GC domain was lower than that of human GC-C, and allosteric regulation of the receptor by ATP binding to the intracellular kinase-homology domain also differed. These altered properties are reflected in the high concentrations of ligands required to elicit signaling responses in the mouse gut in preclinical models and the specificity of a GC inhibitor towards human GC-C. Therefore, our studies identify considerations in using the murine model to test molecules for therapeutic purposes that work as either agonists or antagonists of GC-C, and vaccines for the bacterial heat-stable enterotoxin that causes watery diarrhea in humans.

Inhibition of Heat-Stable Toxin-Induced Intestinal Salt and Water Secretion by a Novel Class of Guanylyl Cyclase C Inhibitors.

BACKGROUND: Many enterotoxigenic Escherichia coli strains produce the heat-stable toxin, STa, which, by activation of the intestinal receptor-enzyme guanylyl cyclase (GC) C, triggers an acute, watery diarrhea. We set out to identify GCC inhibitors that may be of benefit for the treatment of infectious diarrheal disease. METHODS: Compounds that inhibit STa-induced cyclic guanosine 3',5'-monophosphate (cGMP) production were selected by performing cyclase assays on cells and membranes containing GCC, or the related GCA. The effect of leads on STa/GCC-dependent activation of the cystic fibrosis transmembrane conductance regulator anion channel was investigated in T84 cells, and in porcine and human intestinal tissue. Their effect on STa-provoked fluid transport was assessed in ligated intestinal loops in piglets. RESULTS: Four N-2-(propylamino)-6-phenylpyrimidin-4-one-substituted piperidines were shown to inhibit GCC-mediated cellular cGMP production. The half maximal inhibitory concentrations were ≤ 5 × 10(-7) mol/L, whereas they were >10 times higher for GCA. In T84 monolayers, these leads blocked STa/GCC-dependent, but not forskolin/adenylyl cyclase-dependent, cystic fibrosis transmembrane conductance regulator activity. GCC inhibition reduced STa-provoked anion secretion in pig jejunal tissue, and fluid retention and cGMP levels in STa-exposed loops. These GCC inhibitors blocked STa-provoked anion secretion in rectal biopsy specimens. CONCLUSIONS: We have identified a novel class of GCC inhibitors that may form the basis for development of future therapeutics for (infectious) diarrheal disease.

Linaclotide-a novel secretagogue in the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation.

Irritable bowel syndrome with constipation (IBS-C) and chronic idiopathic constipation (CIC) are highly prevalent gastrointestinal disorders. Traditional symptoms based therapies had somewhat limited success and efficacy in addressing the disorders. Recently, linaclotide emerged as novel peptide capable of improving abdominal symptoms in patients suffering from IBS-C and CIC. Guanylate cyclase C (GC-C) receptor a multi domain protein, found to be molecular target for linaclotide which acts by activating GC-C receptor on the apical surface of intestinal epithelial cells. Binding of linaclotide to GC-C receptor triggers the elevation of second messenger cGMP that elicits fluid secretion into intestinal cells which play a critical role in maintaining homeostasis through cystic fibrosis transmembrane conductance regulator (CFTR). Data from Phase II and III clinical trials demonstrated that linaclotide seems to produce a statistically significant increase in stool frequency, improved straining, decreased abdominal pain and discomfort.

Pilot study on the effect of linaclotide in patients with chronic constipation.

OBJECTIVES: Chronic constipation is a common gastrointestinal disorder with limited treatment options. Oral administration of linaclotide, a novel peptide agonist of guanylate cyclase-C receptors, has been shown in animal studies to stimulate intestinal fluid secretion and transit. In Phase 1 studies in healthy human volunteers, linaclotide was well-tolerated, increased bowel movement frequency, and loosened stool consistency. METHODS: This randomized, double-blind, placebo-controlled pilot study evaluated the safety, tolerability, and exploratory efficacy of oral linaclotide in 42 patients with chronic constipation. Patients were randomized to linaclotide (100, 300, or 1,000 microg) or placebo once daily for 2 weeks. Bowel habits (stool frequency, consistency, straining, completeness of evacuation) and degree of abdominal discomfort were monitored daily using an interactive voice response system. Patient-reported outcomes of severity of constipation and overall relief were evaluated weekly. RESULTS: Linaclotide treatment produced dose-dependent increases from the pretreatment baseline values in weekly complete spontaneous bowel movement frequency (range: 2.2-3.2), stool consistency scores (range: 1.1-2.6, 7-point scale), and straining scores (range: 0.4-1.5, 7-point scale); corresponding placebo increases were 1.3, 0.4, 0.4, respectively. Clinical improvements were also demonstrated in abdominal discomfort, severity of constipation, and overall relief. Compared to placebo, linaclotide 100 microg/day significantly increased spontaneous bowel movement frequency, and linaclotide 1,000 microg/day significantly improved stool consistency (P<0.05). Adverse events were primarily gastrointestinal, with diarrhea being the most common. CONCLUSIONS: In this pilot study, linaclotide treatment improved bowel habits and symptoms of patients with chronic constipation. Further randomized controlled trials are warranted.