1. Volume 132, Issue 4, Pages 1234-1244 (April 2007)
Lectin Conjugates as Potent, Nonabsorbable CFTR Inhibitors for Reducing Intestinal Fluid Secretion in Cholera 
N.D. Sonawane, Dan Zhao, Olga Zegarra–Moran, Luis J.V. Galietta, A.S. Verkman 
Gastroenterology 
Volume 132, Issue 4, Pages (April 2007)
DOI: /j.gastro
Copyright © 2007 AGA Institute Terms and Conditions

2. Figure 1
Lectin-based CFTR inhibitors. (A) Structures of malonic acid hydrazide MalH-(PEG)1, glycine hydrazide GlyH-101, DIDS, and CFTRinh-172. (B) Synthesis of “sticky,” nonabsorbable MalH-lectin CFTR inhibitor. MalH-DIDS was conjugated to lectins and albumin to produce MalH-ConA, MalH-wheat, MalH-TL, MalH-aspargus, and MalH-albumin. Reaction conditions: (a) TEA, DMSO, RT, 24 hours, 59%; (b) 50 mmol/L carbonate buffer, pH 8-9, RT, 30 minutes. (C) Inhibition of apical membrane chloride current in FRT epithelial cells expressing human wild-type CFTR. CFTR was stimulated by 20 μmol/L forskolin (forsk). Increasing concentrations were added of MalH-ConA, MalH-wheat and ConA (left) and MalH-DIDS and MalH-albumin (right).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

3. Figure 2
Carbohydrate specificity of MalH-ConA. (A) Effect of unconjugated ConA on inhibition potency of MalH-ConA. (left) Inhibition of apical membrane chloride current by MalH-ConA after CFTR stimulation, in the absence vs presence of 5 μmol/L Con A. (right) Concentration-inhibition data for MalH-DIDS in the presence (filled triangle) vs absence of (open triangle) of 5 μmol/L ConA; data also shown for MalH-ConA in the absence (filled circles) or presence (open circles) of 5 μmol/L ConA. (B) (left) Inhibition of chloride current by MalH-ConA, in the presence of 20 or 200 mmol/L mannose. (right) Concentration-inhibition data for MalH-ConA in the presence of mannose (200 mmol/L, open circles) and MalH-DIDS (with/without 200 mmol/L mannose, closed and open triangles).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
MalH-lectin denaturation reduces CFTR-inhibition potency. (A) MalH-ConA was denatured by acidification (incubation at pH 5.2 or 1.5, top), heating (incubation at 100°C for 5 minutes, middle), or enzymatic digestion (incubation with pronase at 37°C for 6 hours, bottom) before current measurements. Also shown is effect of inclusion of EDTA in buffer at the time of current measurements (bottom). (B) (top) Concentration-inhibition data for MalH-ConA at pH 5.2 (open circles) and 1.5 (open triangles) and for MalH-DIDS (closed triangles, pH 1.5). (middle) Data for heat-treated MalH-DIDS (closed triangles) and MalH-ConA (open circles). (bottom) Data for MalH-ConA in the presence of EDTA (open triangles), and after pronase digestion (open circles), and MalH-DIDS after treatment with pronase (closed triangles). Mean ± SE for measurements on 3–8 cell cultures.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

5. Figure 4
Electrophysiological analysis of CFTR inhibition by MalH-lectin. (A) Representative whole-cell membrane currents (Im) from a CFTR-expressing FRT cell. Each panel shows superimposed membrane currents induced at different membrane potentials (from –100 to +100 mV) in 20-mV steps. Currents were recorded under resting conditions (top), after CFTR activation with 20 μmol/L forskolin (middle) and after the addition of 250 nmol/L MalH-ConA (bottom). (inset) Comparison of kinetics of block by MalH-ConA (250 nmol/L) versus GlyH-101 (5 μmol/L) after stepping the membrane potential to +80 mV. (B) Current-voltage relationships from experiments as in A. (C) Outside-out patch recording showing block of CFTR single-channel currents by 100 nmol/L MalH-ConA. Membrane potential was +80 mV. CFTR was activated by PKA/ATP.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

6. Figure 5
Slow washout and reversibility of MalH-ConA. (A) Apical membrane current measured before and after washout of MalH-DIDS (left) and MalH-ConA (right). (B) Washout of fluorescently labeled MalH-ConA. Cells incubated with MalH-TMR-ConA at 4°C for 3 minutes and washed with saline for 5 minutes (top left) or 30 minutes (top right) or with 200 mmol/L mannose (lower left). “Mannose” indicates labeling of cells in presence of mannose. (bottom) Relative cell-associated TMR fluorescence at indicated times after washout by PBS or mannose (20 mmol/L) and in which cells were preincubated with ConA or mannose (SE, 4 cultures per condition, *P < .05). (C) Inhibitor washout in a suckling mouse model of cholera. Suckling mice were gavaged with control vehicle (left) or with equifluorescent TMR-MalH-ConA (middle) or TMR-dextran (right). Whole-body fluorescence images were collected at indicated times. Photographs typical of 6 mice studied per group.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

7. Figure 6
Antisecretory efficacy of MalH-ConA. (A) Inhibition of short-circuit current in (nonpermeabilized) T84 cells by MalH-ConA and MalH-wheat after CFTR stimulation. Amiloride and forskolin were added as indicated. (B) Intestinal fluid accumulation at 6 h in closed midjejunal loops in mice (SE, 6–8 loops studied per condition, *P < .05, analysis of variance). (C) Improved survival of suckling mice (30 mice per group) after gavage with cholera toxin without vs with MalH-ConA (125 pmol, P = .0015, log-rank test, top) and MalH-wheat (200 pmol, P = .0012, bottom). “Control” refers to identically processed mice not given cholera toxin or MalH-lectins. (D) Proposed mechanism for increased affinity and slowed intestinal washout of MalH-lectin conjugates. MalH-lectin conjugate depicted with flexible spacer and sugar binding sites, 1 per each lectin monomer of the tetramer. Lectin binding to sugar epitopes within the cell-surface glycocalyx facilitates inhibitor entrapment and increased local concentration in the vicinity of the target CFTR.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions