title: Lectin conjugates as potent, nonabsorbable CFTR inhibitors for reducing intestinal fluid secretion in cholera.
abstract: OBJECTIVE
  Inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are predicted to prevent intestinal fluid secretion in cholera. We previously discovered low- affinity glycine hydrazide (GlyH) CFTR inhibitors that block CFTR at its external pore. The goal of this study was to develop potent CFTR inhibitors that are minimally absorbed and washed out of the intestinal lumen for application as antisecretory agents in cholera.
  
  
  METHODS
  GlyH analogs (malonic hydrazides, MalH) were chemically conjugated to various lectins ("MalH-lectin") and purified. CFTR inhibition potency was measured by short-circuit current analysis, mechanism of action by patch-clamp, and antidiarrheal efficacy in closed-loop and suckling mouse models.
  
  
  RESULTS
  By lectin conjugation, we improved CFTR inhibitory potency by approximately 100-fold (to 50 nmol/L) and retarded washout. High-affinity CFTR inhibition was abolished by MalH-lectin heat denaturation, protease digestion, or competition by mannose or unconjugated lectin. Patch-clamp analysis indicated CFTR inhibition by an external pore occlusion mechanism. Fluorescently labeled MalH-lectin remained membrane bound for >6 hours after washout, whereas washout occurred in a few minutes without the lectin. MalH-ConA and MalH-wheat (IC50 50-100 pmol) blocked cholera toxin-induced intestinal fluid secretion in closed intestinal loops in mice and greatly reduced mortality in a suckling mouse model of cholera.
  
  
  CONCLUSIONS
  The high potency of MalH-lectin conjugates results from "anchoring" the CFTR-blocking MalH to cell surface carbohydrates by the lectin. The high-affinity, slow washout, and external site of action of the MalH-lectin conjugates support their further development as antisecretory drugs for enterotoxin-mediated secretory diarrheas.
authors.affiliation: Departments of Medicine and Physiology, University of California, San Francisco, California, USA.
authors.forename: N D
authors.initials: ND
authors.lastname: Sonawane
authors.forename: Dan
authors.initials: D
authors.lastname: Zhao
authors.forename: Olga
authors.initials: O
authors.lastname: Zegarra-Moran
authors.forename: Luis J V
authors.initials: LJ
authors.lastname: Galietta
authors.forename: A S
authors.initials: AS
authors.lastname: Verkman
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002771:Cholera / Q000150:complications / Q000188:drug therapy* / Q000378:metabolism; D002772:Cholera Toxin / Q000633:toxicity; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors* / Q000378:metabolism; D003681:Dehydration / Q000188:drug therapy* / Q000209:etiology / Q000378:metabolism; D004195:Disease Models, Animal; D045604:Extracellular Fluid / Q000378:metabolism*; D007408:Intestinal Absorption / Q000187:drug effects; D007421:Intestine, Small / Q000187:drug effects / Q000378:metabolism*; D037102:Lectins / Q000627:therapeutic use*; D051379:Mice; D008807:Mice, Inbred BALB C; D018408:Patch-Clamp Techniques; D011042:Poisons / Q000633:toxicity; D051381:Rats; D011916:Rats, Inbred F344
chemical_list: D037102:Lectins; D011042:Poisons; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D002772:Cholera Toxin