therapeutically targeting guanylate cyclase‐c...

ORIGINAL ARTICLE

Therapeutically targeting guanylate cyclase-C:computational modeling of plecanatide, a uroguanylinanalogAndrea Brancale1 , Kunwar Shailubhai2, Salvatore Ferla1, Antonio Ricci1, Marcella Bassetto1 &Gary S Jacob2

1School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, United Kingdom2Synergy Pharmaceuticals, New York, New York

Keywords

guanylate Cyclase-C, linaclotide, molecular

dynamics, plecanatide, uroguanylin

Correspondence

Andrea Brancale, Cardiff School of Pharmacy

and Pharmaceutical Sciences, Cardiff

University, Redwood Building, King Edward

VII Avenue, Cardiff, CF10 3NB.

Tel: +44 (0)29 2087 4485;

Fax: +44(29) 2087 4149;

E-mail: [email protected]

Funding Information

Financial Support for this study provided by

Synergy Pharmaceuticals Inc. We also

acknowledge the support of the Life Science

Research Network Wales grant #

NRNPGSep14008, an initiative funded

through the Welsh Government’s Ser Cymru

program.

Received: 15 August 2016; Revised: 23

November 2016; Accepted: 30 November

2016

Pharma Res Per, 5(2), 2017, e00295,

doi: 10.1002/prp2.295

doi: 10.1002/prp2.295

Abstract

Plecanatide is a recently developed guanylate cyclase-C (GC-C) agonist and the

first uroguanylin analog designed to treat chronic idiopathic constipation (CIC)

and irritable bowel syndrome with constipation (IBS-C). GC-C receptors are

found across the length of the intestines and are thought to play a key role in

fluid regulation and electrolyte balance. Ligands of the GC-C receptor include

endogenous agonists, uroguanylin and guanylin, as well as diarrheagenic,

Escherichia coli heat-stable enterotoxins (ST). Plecanatide mimics uroguanylin

in its 2 disulfide-bond structure and in its ability to activate GC-Cs in a pH-

dependent manner, a feature associated with the presence of acid-sensing resi-

dues (Asp2 and Glu3). Linaclotide, a synthetic analog of STh (a 19 amino acid

member of ST family), contains the enterotoxin’s key structural elements,

including the presence of three disulfide bonds. Linaclotide, like STh, activates

GC-Cs in a pH-independent manner due to the absence of pH-sensing residues.

In this study, molecular dynamics simulations compared the stability of pleca-

natide and linaclotide to STh. Three-dimensional structures of plecanatide at

various protonation states (pH 2.0, 5.0, and 7.0) were simulated with GRO-

MACS software. Deviations from ideal binding conformations were quantified

using root mean square deviation values. Simulations of linaclotide revealed a

rigid conformer most similar to STh. Plecanatide simulations retained the flexi-

ble, pH-dependent structure of uroguanylin. The most active conformers of ple-

canatide were found at pH 5.0, which is the pH found in the proximal small

intestine. GC-C receptor activation in this region would stimulate intraluminal

fluid secretion, potentially relieving symptoms associated with CIC and IBS-C.

Abbreviations

CIC, chronic idiopathic constipation; FGID, functional gastrointestinal disorder;

GC-C, guanylate cyclase-C; GI tract, gastrointestinal tract; IBS-C, irritable bowel

syndrome with constipation; RMSD, root mean square deviation; ST, family of heat

stable enterotoxin produced by enterotoxigenic Escherichia coli that include STh

and STp; STh, 19 amino acid member of ST family.

Introduction

Chronic idiopathic constipation (CIC) and irritable bowel

syndrome with constipation (IBS-C) are two of the most

common conditions affecting the gastrointestinal (GI)

tract, creating a burden on healthcare resources and lead-

ing to significant negative impact on quality of life (Hei-

delbaugh et al. 2015). These disorders are characterized

by diminished stool frequency, straining and abdominal

pain (IBS-C) or discomfort (CIC). CIC alone affects 14%

ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,

British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.

This is an open access article under the terms of the Creative Commons Attribution License,

which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

2017 | Vol. 5 | Iss. 2 | e00295

http://orcid.org/0000-0002-9728-3419

http://orcid.org/0000-0002-9728-3419

http://orcid.org/0000-0002-9728-3419

info:doi/10.1002/prp2.295

http://creativecommons.org/licenses/by/4.0/

of the North American population, is challenging to treat

and poses a significant burden on health resources

(Suares and Ford 2011).

Guanylate cyclase-C (GC-C) receptors play a crucial

role in the maintenance of normal bowel function and

thus have potential as a target for pharmaceutical inter-

vention in to treat numerous functional gastrointestinal

disorders. The GC-C receptor is a membrane-spanning

protein uniformly expressed along the epithelial brush

border throughout the intestine (Forte 1999). Recently

plecanatide and linaclotide have been developed for the

treatment of two of these disorders, CIC and IBS-C

(Shailubhai et al. 2013).

The GC-C receptor is activated by its endogenous pep-

tides uroguanylin and guanylin that differentially bind to

the receptor in the varying pH environments found along

the GI tract (Fan et al. 1997). Uroguanylin is primarily

expressed and preferentially binds GC-C receptors in the

slightly acidic (pH 5-6) regions of the duodenum and

jejunum (Forte 1999). Guanylin is primarily expressed in

the ileum and colon, activating GC-C receptors under

more basic conditions (pH 7 -8) (Kita et al. 1994). Bind-

ing of uroguanylin or guanylin to the GC-C receptor ini-

tiates a signaling cascade leading to accumulation of

intracellular cyclic guanosine monophosphate (cGMP)

(Vaandrager et al. 1997), which helps maintain fluid and

electrolyte balance, promotes visceral analgesia, and

reduces inflammation in the GI tract (Hughes et al. 1978;

Pitari 2013; Shailubhai et al. 2015; Hanning et al. 2014).

The overlapping yet distinct activities of guanylin and

uroguanylin suggest that the tight and tunable regulation

of GC-C receptors is essential for proper GI function, a

feature that becomes readily apparent by the conse-

quences of dysfunctional GC-C activity (Whitaker et al.

1997). Overactivation of GC-C receptors by the E. coli

enterotoxin (STh) triggers an uncontrolled release of elec-

trolytes and water into the intestinal lumen resulting in

diarrhea (Brierley 2012).

Studies have shown STh to be 10 times more potent than

uroguanylin and 100 times more potent than guanylin in

binding to GC-C receptors (Hamra et al. 1993). The X-ray

structure of STh reveals that the molecule is locked into a

constitutively active, right-handed spiral formation stabi-

lized by three intrachain disulfide bridges in a 1-4/2-5/3-6

pattern (Gariepy et al. 1987; Ozaki et al. 1991; Shimonishi

et al. 1987). Unlike STh, the endogenous peptides have only

two disulfide bridges which likely results in their improved

flexibility; Klodt et al. (1997). The flexibility afforded by

absence of a third disulfide bridge allows uroguanylin and

guanylin to adopt two topological isoforms (A and B) of

which only the A form is biologically active (Marx et al.

1998; Skelton et al. 1994). The pH-dependent activity of

uroguanylin is linked to two charged acid-sensing aspartic

acid residues on its N-terminus (Hamra et al. 1997). The

absence of these residues within STh allows it to bypass pH

checkpoints governing GC-C receptor activation, allowing

for supraphysiological activation of GC-C along the length

of the small intestine and colon (Hamra et al. 1997).

Pharmacologic agonists that mimic the activity of

known GC-C agonists have been developed to treat

patients with CIC and IBS-C. Linaclotide, a synthetic ana-

log of STh, is available for the treatment of CIC and IBS-

C (Lembo et al. 2011). Like STh, linaclotide has three

disulfide bonds and demonstrates pH-independent activa-

tion of GC-C receptors (Fig. 1) (Busby et al. 2010).

Plecanatide is a recently developed GC-C agonist and

uroguanylin analog that is currently in Phase 3 trials for

CIC and IBS-C (Synergy Pharmaceuticals Inc 2015a,b).

Plecanatide is an orally administered, pH-dependent ago-

nist of the GC-C receptor that shares the structural and

physiological characteristics of uroguanylin. Plecanatide

has two disulfide bonds, similar to uroguanylin, and con-

tains two acidic N-terminal amino acids allowing the two

molecules to maintain the same pH-dependent binding

Figure 1. Amino acid structures of guanylate cyclase-C receptor

agonists examined in this study. Synthetic analogs linaclotide and

plecanatide share similar amino acid sequences with GC-C agonists

STh and uroguanylin, respectively. Plecanatide, like uroguanylin

contains two pH-sensing residues on its N-terminus. The pH-sensing

residue (aspartatic acid, D) of uroguanylin is replaced with another

pH-sensing residue (glutamic acid, E) in plecanatide (green). In this

study, the pH-sensing aspartic acid and glutamic acid residues of

plecanatide were differentially protonated to reflect pH values 2.0

(Asp2, Glu3), 5.0 (Asp2-,Glu3; Asp2, Glu3-) & ≥7.0 (Asp2-, Glu3-).

Simulations of the crystal structure of STh used a truncated version of

the full toxin, comprised of residues 5–17 of the full heat-stable

enterotoxin protein representing the core bioactive pharmacophore of

peptide. (A). STh and linaclotide both lack pH-sensing residues on

their N-terminal ends and are stabilized into a constitutively active

conformer by the presence of 3 disulfide bonds. Structurally, the

peptides differs by the replacement of leucine (L) in STh with tyrosine

(Y*) in linaclotide.

2017 | Vol. 5 | Iss. 2 | e00295



Computational Modeling of Plecanatide, a Uroguanylin Analog B. Andrea et al.

characteristics (Fig. 1) (Shailubhai et al. 2013). Given the

experimental challenges of studying the unique character-

istics of its pH-sensitive structure, computational methods

were employed to characterize behavior of plecanatide at

various pH states.

Molecular dynamics (MD) simulations compared the

flexibility and conformation of plecanatide and linaclotide.

As expected, linaclotide was shown to adopt rigid confor-

mations, which do not deviate significantly from the struc-

ture of STh. In contrast, plecanatide showed greater

flexibility and an ability to adopt several conformations

which vary in response to pH values (2.0, 5.0 and 7.0).

Active plecanatide conformations were more similar to

uroguanylin than to the STh peptide, suggesting that pleca-

natide has a similar pH-dependent activity profile as uro-

guanylin and that plecanatide’s activity can be differentially

regulated in the GI tract, with higher activity in the more

acidic proximal small intestine and lower activity in the

more basic distal small intestine and colon.

Materials and Methods

Peptide preparation

The NMR structures of uroguanylin (PDB ID: 1UYA)

and the crystal structure of STa (PDB ID: 1ETN)

(Fig. S1) were used as starting points for the MD simula-

tions (Ozaki et al. 1991). The STa family of heat stable

peptides includes STh and STp (Nataro and Kaper 1998).

The series of experiments used in this study used the STh

sequence as reference.

Structural models of plecanatide, linaclotide, and STh

were built by appropriately modifying the uroguanylin and

STa sequences, respectively, using the builder tools in molec-

ular operating environment [(MOE 2015.4) www.chemc

omp.com]. (Molecular Operating Environment 2015).

Simulations and root mean standard deviation (RMSD)

values of linaclotide and each configuration of plecanatide

were compared to the enteric pathogen, STh, which was

selected as the reference peptide because of its enhanced

affinity for the GC-C receptor compared to other ago-

nists. The structural rigidity of STh confers one main

conformation that would bind the receptor. Similarities

to this one conformer would reflect a drugs ability to

activate GC-C.

MD simulations

All MD simulations of plecanatide, STh, and linaclotide

were performed and analyzed using the GROMACS 4.5

simulation package (Hess et al. 2008). MD simulations of

the structure of STh used residues 6–18 of the full heat-

stable enterotoxin protein (C-C-E-L-C-C-N-P-A-C-T-G-C),

considered to be the pharmacophore of the molecule

required for maximum biological activity (Ozaki et al.

1991; Yoshimura et al. 1985).

Four protonation states of plecanatide were modeled

by placing the appropriate charge on Asp2 and Glu3,

reflecting the most abundant species at three pH environ-

ments: pH 2.0 (Asp2, Glu3), pH 5.0 (Asp2-, Glu3; Asp2,

Glu3-), and pH 7.0 (Asp2-, Glu3-) (Fig. 1 circle). The ini-

tial structure of each peptide was placed in a cubic box

with TIP 3P water and energy minimized using a Steepest

Descent Minimization Algorithm. The system was equili-

brated via a 50 ps MD simulation at 310 K in a NVT

canonical environment followed by an additional 50 ps

simulation at constant pressure of 1 atm (NPT). After the

equilibration phases, a 500 ns MD simulations were per-

formed at constant temperature (310 K) and pressure

with a time step of 2 fs. The system energy and peptide

spatial coordinates (trajectory file) were stored every

300 ps for further studies. All MD simulations were run

in triplicate for each peptide.

After removing the first 100 ns, considered as system

stabilization time, the remaining 400 ns of the MD trajec-

tory of every single run for each group of triplicate exper-

iments were combined using the tricat function. The

combined trajectories (3999 frames) were examined using

the g-cluster function, setting gromos as the clustering

method with an RMSD cut-off of 0.1 nm. The different

structural cluster groups were obtained as a pdb file.

Cluster groups representing at least 10% of the total pop-

ulation for each peptide were selected as the most repre-

sentative structure for that peptide.

RMSD comparisons

The representative cluster conformations of the different

peptides were used for the RMSD comparison against the

main conformation of the STh cluster.

RMSD comparisons were performed using MOE 2015.4

with the major STh cluster structure serving as a reference

for the superimposition of other peptide conformations.

Results

MD clustering

Table 1 shows the results obtained from the structural

cluster calculations. From this data it is possible to appre-

ciate how flexible plecanatide is, compared to STh and

Linaclotide. The latter peptides generated more populated

cluster than any of the plecanatide forms. RMSD analysis

of each MD simulations calculated against the most rep-

resentative clusters also confirm this observation

(Fig. S1).

ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.

2017 | Vol. 5 | Iss. 2 | e00295

B. Andrea et al. Computational Modeling of Plecanatide, a Uroguanylin Analog

http://www.chemcomp.com

http://www.chemcomp.com

MD simulations of STh and linaclotide

In addition to providing information on structure, the

simulations also provided information on the degree of

internal rigidity and flexibility within the peptide. This

can be observed in the MD simulations shown in Fig-

ure 2A, in which overlapping snapshots of STh show very

little deviation. The fact that the snapshots show a high

degree of overlap across all rounds of simulations indi-

cates that STh is a fairly rigid molecule with little internal

flexibility. This constrained geometry is thought to be

established by the three disulfide bonds of the peptide

leading to a structural rigidity and high binding affinity

of STh for the GC-C receptor (Ozaki et al. 1991).

MD simulations of linaclotide, which differs from the

STh peptide used in the simulations by one amino acid

substitution and an additional residue at the C-terminus,

also reveal a rigid molecule that adopts a similar confor-

mation as STh (Fig. 2B). The rigidity of this peptide was

also confirmed by a RSM fluctuation analysis of the MD

trajectory (Supplemental Information, Fig. S2). Some

variability can be seen within the C-termini of the pep-

tides, likely due to the extra amino acid in linaclotide

compared to STh. Moreover, the conformation of the

interaction loops (regions binding the GC-C receptor) is

homologous between the two molecules (Fig. 2B *)(Ozaki et al. 1991).

MD simulations of plecanatide

MD simulations of plecanatide were conducted by alter-

ing the amino acid sequence of the NMR structure of

uroguanylin (Marx et al. 1998). Because pH has been

shown to alter the ability of uroguanylin to activate GC-C

receptors, simulations were conducted on the four ioniza-

tion states of plecanatide’s structure, reflecting three dif-

ferent pH values, by altering the protonation states of

Asp2 and Glu3 residues. It should be noted that pleca-

natide contains an additional pH-sensitive residue, Glu5.

However, unlike Glu3, its side chain is oriented away

from the interaction loop and, given its position between

the two disulfide bonds, Glu5 does not have the confor-

mational freedom to affect the orientation of the loop

itself. Furthermore, this specific residue is highly con-

served across the whole range of GC-C binding peptides,

and includes STh and linaclotide which are not affected

by pH variations (Busby et al. 2010). For these reasons,

the protonation state of Glu5 should not affect the activ-

ity of uroguanylin and plecanatide.

To represent plecanatide at pH 5.0, which corresponds

to the pH of the duodenum and proximal jejunum, two

Table 1. Representative cluster analysis.

Compound and

test condition

Cluster

size1% On the

total frames2Average

RMSD (�A) � SD3

STh 2273 56 0.1252 � 0.0821

Linaclotide 3592 89 0.0715 � 0.0421

Plecanatide –

pH>7.0

1108 28 0.1827 � 0.0862

Plecanatide –

pH 5.0 (Asp-/Glu)4524

497

446

13

12

11

0.3208 � 0.1325

0.2425 � 0.1044

0.1252 � 0.1107

Plecanatide –

pH 5.0 (Asp/Glu-)

610 15 0.2885 � 0.1363

Plecanatide – pH<2.0 709 18 0.2115 � 0.0825

1The cluster size refers to the number of frames that are forming the

cluster. The total number of frames is 3999.2The % is calculated on the total number of frames.3The average RMSD is calculated against the most representative clus-

ter conformation for each peptide.4Three representative clusters were obtained for this peptide.

Figure 2. MD simulations of STh and linaclotide. (A) Overlapping snapshots of STh from MD simulations reveal that the peptide adopts a single

stable structure with little flexibility. (B) Superimposition of representative structures from the STh and linaclotide. Variations between structures at

the C-terminus reflects changes in conformation induced by the additional tyrosine of linaclotide. Simulations reveal that linaclotide adopts a

similar conformation as STh especially so within the region of the GC-C interaction loop. (*). MD, Molecular dynamics.

2017 | Vol. 5 | Iss. 2 | e00295




protonation configurations were analyzed. In one, Asp2 is

protonated (Asp/Glu-), whereas in the other, Glu3 is pro-

tonated (Asp-/Glu). These ionization states were based on

the consideration that, as these residues are on the flexible

N-terminus and exposed to solvent, their pKa values

would be between 3.5 and 4.5 (values dependent on the

input peptide conformation as calculated on http://bio

physics.cs.vt.edu/H++, version 3.2) (Anandakrishnan et al.

2012); hence, the monoprotonated states would likely be

present at pH 5.0. Simulations of the two protonation

states indicate that plecanatide is flexible at this pH and

can adopt several conformations. Figures 3A–C show an

overlay of the three predominant Asp-/Glu conformations

of plecanatide and STh, and Figure 3D shows the pre-

dominant Asp/Glu- conformation of plecanatide and STh.

In two of these structures (Fig. 3A and D), the interaction

loop (*) of plecanatide overlays well with that of STh,

indicating that these two conformations of plecanatide

are capable of binding to and subsequently activating GC-

C receptors.

Simulations of the double protonated form of pleca-

natide (Asp/Glu) were conducted to assess the structure

and dynamics of the peptide at pH 2.0. The plecanatide

conformations observed at this pH differ from those of

STh (Fig. 3E). Based on these results, plecanatide is unli-

kely to adopt a conformation capable of binding to GC-C

at this highly acidic pH level, a feature which would

mimic uroguanylin’s inability to activate GC-C receptors

at this pH.

Simulations of the double negative form of plecanatide

(Asp-/Glu-), which represent the protonation state

observed at pH > 7.0, reveal a single predominant pleca-

natide structure (Fig. 3F). The portion of the peptide that

interacts with the GC-C receptor adopts a different con-

formation in plecanatide than in STh indicating dimin-

ished activity of the molecule at this pH value.

Interestingly, in the Asp/Glu- ionization state of pleca-

natide at pH 5, an interaction occurred between the

negatively charged acidic side chain of Glu3 residue in

the N-terminus and the positively charged side chain of

Asn9 in the interaction loop (Fig. 4). This interaction

between the Glu3 residue of the N-terminus and the Asn9

residue of the interaction loop seems to stabilize pleca-

natide in its most active conformation at pH 5. This

Figure 3. Overlapping snapshots of STh and plecanatide at pH values 5.0 (A–D), 2.0 (E) and >7.0 (F) using MD simulations. (A–C) Overlay of the

three predominant Asp-/Glu conformations of plecanatide and STh at pH 5.0 (RMSD values – A: 2.38 �A; B:2.48 �A; C:3.44 �A). (D) The

predominant Asp/Glu- conformation of plecanatide and STh at pH 5.0 (RMSD value–1.93 �A). The overlapping conformation of the interaction

loops (*) in A and D suggest these are active forms of plecanatide able to bind to and stimulate GC-C receptors. (E) Plecanatide conformations at

pH 2.0 Asp/Glu differ from those of STh with no overlap of interaction loop (RMSD value–3.45 �A). (F) Simulations of the double negative form of

plecanatide, Asp-/Glu-, representing the protonation state at pH > 7.0, reveal a single plecanatide structure that has a minimal overlap of the

interaction loop (RMSD value – 2.54 �A). MD, Molecular dynamics.


2017 | Vol. 5 | Iss. 2 | e00295


http://biophysics.cs.vt.edu/H

http://biophysics.cs.vt.edu/H

interaction was not observed at other pH values nor is it

expected to occur with uroguanylin as the Asp3 amino

acid in uroguanylin would not be of sufficient length to

interact with the Asn9 residue in its interaction loop.

Overall, the highly flexible behavior of plecanatide is

very similar to the one observed with the parent peptide

uroguanylin (Fig. 5). Indeed, the NMR structures avail-

able for the latter peptide show a high degree of confor-

mational variability that closely mimics plecanatide. The

RMS Fluctuation analysis on Plecanatide also confirms

this flexibility (Fig. S2). The interaction loop of the two

peptides overlaps generally very well. Some variability is

observed in the N-terminus, where the substitution of

Asp3 for Glu3 is present.

In summary, if we consider all simulation sets at pH

2.0, 5.0, and 7.0, we can observe that plecanatide has the

required flexibility at these pH values to switch between

numerous conformations. The most active forms of pleca-

natide were found with the two ionization states represen-

tative of pH 5.0 as revealed by the similarity of their

interaction loops with the corresponding regions in uro-

guanylin at pH 5 (Fig. 5) and STh (Fig. 3A and F). These

results are consistent with previous studies reporting that

plecanatide was designed to be more active at slightly

acidic pH values, mimicking the pH-sensitive behavior of

uroguanylin (Shailubhai et al. 2013).

Comparison of MD simulations

RMSD comparisons of the different peptides analyzed

were used to quantify the similarities between the struc-

tures. The STh structure was used as a reference and the

conformations of the different peptides were superim-

posed on the STh molecule to evaluate their similarity to

the prototype of the active peptide, with lower RMSD

values indicating more similarity. Table 2 shows the

RMSD value for each peptide, considering all the repre-

sentative clusters. With an RMSD of 1.28 �A, the structure

of linaclotide is more similar to STh than to uroguanylin

or to any of the plecanatide variants. The RMSD values

of the different plecanatide conformations show how flex-

ible the peptide is. Interestingly, plecanatide presents

some conformations that are very close to the reference

STh (RMSD <2 �A), suggesting a peak activity for pleca-

natide at this pH. Additionally, RMSD calculations reveal

Figure 4. Structure of plecanatide at pH 5.0, in the Asp/Glu-

ionization state. Interaction (*) between the negative charge of Glu3

residue on the pH-sensitive N-terminus and Asn9 residue in the

interaction loop potentially serves to stabilize this active conformation

of plecanatide.

Figure 5. Overlay of the most active conformation of plecanatide,

Asp/Glu-, (pH 5.0), with the active “A” NMR structure of uroguanylin.

The interaction loop of the two peptides overlaps generally very well

(*). Some variability is observed in the N-terminus, where the

substitution Asp3/Glu3 is present.

Table 2. RMSD comparison between STh and the most representa-

tive clusters of the different peptides simulated.

Compound and Test Condition RMSD–�A

Linaclotide 1.28

Uroguanylin 1.2–2.341

Plecanatide - pH>7.0 2.54

Plecanatide – pH 5.0 (Asp-/Glu) 3.44, 2.48, 2.382

Plecanatide – pH 5.0 (Asp/Glu-) 1.93

Plecanatide – pH<2.0 3.45

1Range of RMSD values between STh and the 10 NMR conformations

of uroguanylin.2Three representative clusters (>10% – see methods section) were

obtained for this peptide. RMSD comparisons of the different peptides

analyzed were used to quantify the similarities between the struc-

tures. Lower values indicate higher structural similarity with STh refer-

ence molecule.

2017 | Vol. 5 | Iss. 2 | e00295




that these four variants of plecanatide are the closest

structurally to the active A-form of uroguanylin. This is

consistent with the data described above, in which the

negative charge of Glu3 interacts with Asn9 in the inter-

action loop, potentially promoting an active conformation

of plecanatide. These results indicate that, in a manner

similar to uroguanylin, the activity of plecanatide is not

“all or nothing” but tunable based upon the pH of the

environment.

Discussion

Transmembrane GC-C receptors play a critical role in the

regulation of fluid and electrolyte homeostasis within the

GI tract (Brierley 2012; Steinbrecher 2014). This balance

is normally maintained by the pH-dependent activation

of these receptors by the endogenous ligands, uroguany-

lin, and guanylin (Forte 2004). Studies have shown that

these pH environments are maintained at differential val-

ues across the length of the gastrointestinal tract (Daniel

et al. 1985; Whitaker et al. 1997). In the slightly acidic

mucosal environments of the duodenum and proximal

jejunum (pH 5.0-6.0), uroguanylin is 10 times more

potent than in the slightly basic (pH 7.0-8.0) environ-

ments of the ileum and colon (Hamra et al. 1997). In

contrast, guanylin is significantly more potent at binding

GC-C receptors in the ileum and colon (pH 7.0-8.0) than

in the duodenum and proximal jejunum (pH 5.0-6.0),

where it is essentially inactive (Hamra et al. 1997). Struc-

tural shifts induced by pH environments alter the potency

of these ligands, allowing them to cooperatively regulate

GC-C receptor activation (Hamra et al. 1997). Any per-

turbation in the pH-dependent control of GC-C activa-

tion would disrupt the delicate balance of electrolytes and

fluids within the intestines. This disruption could be asso-

ciated with disorders such as obstruction, secretory diar-

rhea and inflammatory bowel disease (Field 2003;

Fiskerstrand et al. 2012).

Based on their physiology, GC-C receptors are a

promising therapeutic target in the treatment of numer-

ous gastrointestinal disorders such as CIC and IBS-C.

Both are common complaints among all age groups and

are associated with a substantial burden on healthcare

resources in the US (Drossman et al. 2009). While the

exact pathologic mechanism of these diseases remains an

area of intense study, the symptoms (i.e. diminished stool

frequency, hard/lumpy stools, straining abdominal symp-

toms) indicate an imbalance in fluid and electrolyte

homeostasis in the GI tract (Steinbrecher 2014).

As an analog of the pathological GC-C agonist STh,

linaclotide maintains many structural features of STh,

including the presence of three disulfide bonds and an

insensitivity to pH. MD simulations in this study show

that the addition of a third intramolecular bond makes

both STh and linaclotide insensitive to MD perturbations

(Ozaki et al. 1991). The structural similarity of these two

molecules is reflected by the low RMSD values of 1.28 �A

for linaclotide. The amino acid substitutions that differen-

tiate linaclotide from STh further enhance the pharma-

cokinetic stability and proteolytic resistance of linaclotide,

allowing it to remain active across a longer portion of the

small intestine (Bharucha and Waldman 2010; Harris and

Crowell 2007). The absence of pH-sensing amino acid

residues would additionally give these molecules maxi-

mum biological activity across the range of pH environ-

ments in the GI tract. This lack of focused areas of

activity may induce excessive fluid secretion and explain

the increased incidence of diarrhea associated with lina-

clotide (Busby and Ortiz 2014; Carpick and Gariepy 1993;

Lembo et al. 2011).

Plecanatide, a GC-C agonist under investigation for

IBS-C and CIC, differs from linaclotide in its amino acid

sequence and number of disulfide bonds. Plecanatide

maintains many of the structural and functional charac-

teristics of endogenous ligand uroguanylin, including the

two disulfide bonds and the two N-terminal pH-sensing

acidic residues, which result in a molecule that can exhi-

bit differential levels of activity based upon the pH of the

environment. As a result, plecanatide has a more targeted

zone of activation, stimulating GC-C receptors under

acidic conditions in a way that is similar to uroguanylin

(Shailubhai et al. 2015).

MD simulations of plecanatide in this study revealed

plecanatide to be a flexible structure that is unlike STh or

linaclotide and one that is able to adopt numerous con-

formations in response to a range of simulated pH envi-

ronments. Optimally active conformations were observed

in pH 5.0 simulations which approximates the pH values

of the duodenum and proximal jejunum. This pattern of

optimal activity is similar to that of uroguanylin, indicat-

ing that plecanatide and uroguanylin are similar in both

structure and function (Shailubhai et al. 2013). In con-

trast, at simulated pH values of 2.0 and 7.0, plecanatide

adopted conformations that would make it less likely to

bind GC-C receptors. Across the range of assessed pH

values, MD simulations and RMSD analysis reveal that

the structure of plecanatide and the NMR structure of

uroguanylin are similar to each other (Fig. 5), and quite

dissimilar to the STh structure (1.93–3.45 �A and 1.20–2.34 �A vs. STh, respectively).

As mentioned previously, the sole difference between

the structures of uroguanylin and plecanatide is the

replacement of an aspartic acid residue (Asp3) with a glu-

tamic acid residue (Glu3) in plecanatide (Fig. 1). Previous

studies have shown that while plecanatide has a similar

pH-dependent affinity for the GC-C receptor, it binds


2017 | Vol. 5 | Iss. 2 | e00295


with superior potency than uroguanylin (Shailubhai et al.

2000, 2015). Computational models in this study revealed

that at pH 5.0, the charged end of the Glu3 residue inter-

acts with the Asn9 residue, potentially serving to stabilize

the structure when it forms its most active conformation.

Interestingly, the corresponding interaction between Asp3

and Asn9 is not present in uroguanylin, probably because

the side chain of Asp3 is not long enough to reach Asn9.

Stabilizing the molecule in this way allows plecanatide to

remain in its active conformer for a longer period of time

at this pH and explain the superior binding profile of ple-

canatide versus uroguanylin witnessed in earlier studies.

Recognizing the acidic-to-basic pH gradient nature of the

GI tract, the simulations in this study suggest that, similar

to uroguanylin, plecanatide would be optimally active in

the more acidic environment that is present in the proximal

small intestine of the GI tract. The ability of plecanatide to

activate GC-C receptors in a targeted manner similar to the

endogenous ligand uroguanylin may help normalize bowel

movements and explain the low levels of adverse events

seen with the drug in early clinical trials (Shailubhai et al.

2013). Understanding the structural mechanisms by which

plecanatide and linaclotide activate GC-C receptors and

respond to pH could shed light on clinical differences

between the two therapeutic options.

Acknowledgements

We wish to acknowledge Medical Dynamics for assistance

in the preparation of this manuscript. We also acknowl-

edge the support of the Life Science Research Network

Wales grant no. NRNPGSep14008, an initiative funded

through the Welsh Government’s Ser Cymru program.

Authorship Contributions

Brancale and Shailubhai participated in research design.

Ferla, Ricci, and Bassetto conducted the experiments.

Brancale and Ferla performed data analysis. Brancale,

Shailubhai, and Jacob wrote or contributed to the writing

of the manuscript.

Disclosures

K Shailubhai and G S Jacob are employees and stockhold-

ers of Synergy Pharmaceuticals.

References

Anandakrishnan R, Aguilar B, Onufriev AV (2012). H++ 3.0:

automating pK prediction and the preparation of bimolecular

structures for atomistic molecular modeling and simulation.

Nucleic Acids Res 40: W573–541.

Bharucha AE, Waldman SA (2010). Taking a lesson from

microbial diarrheagenesis in the management of chronic

constipation. Gastroenterology 138: 813–817.

Brierley SM (2012). Guanylate cyclase-C receptor activation:

unexpected biology. Curr Opin Pharmacol 12: 632–640.

Busby RW, Ortiz S (2014). Clarification of linaclotide

pharmacology presented in a recent clinical study of

plecanatide. Dig Dis Sci 59: 1066–1067.

Busby RW, Bryant AP, Bartolini WP, Cordero EA, Hannig G,

Kessler MM, et al. (2010). Linaclotide, through activation of

guanylate cyclase C, acts locally in the gastrointestinal tract to

elicit enhanced intestinal secretion and transit. Eur J

Pharmacol 649(1–3): 328–335.

Carpick BW, Gariepy J (1993). The Escherichia coli heat-stable

enterotoxin is a long-lived superagonist of guanylin. Infect

Immun 61: 4710–4715.

Daniel H, Neugebauer B, Kratz A, Rehner G (1985).

Localization of acid microclimate along intestinal villi of rat

jejunum. Am J Physiol 248(3 Pt 1): G293–G298.

Drossman DA, Chey WD, Johanson JF, Fass R, Scott C, Panas

R, et al. (2009). Clinical trial: lubiprostone in patients with

constipation-associated irritable bowel syndrome–results oftwo randomized, placebo-controlled studies. Aliment

Pharmacol Ther 29: 329–341.

Fan X, Hamra FK, London RM, Eber SL, Krause WJ, Freeman

RH, et al. (1997). Structure and activity of uroguanylin and

guanylin from the intestine and urine of rats. Am J Physiol

273(5 Pt 1): E957–E964.

Field M (2003). Intestinal ion transport and the

pathophysiology of diarrhea. J Clin Invest 111: 931–943.

Fiskerstrand T, Arshad N, Haukanes BI, Tronstad RR, Pham

KD, Johansson S, et al. (2012). Familial diarrhea syndrome

caused by an activating GUCY2C mutation. N Engl J Med

366: 1586–1595.

Forte LR (1999). Guanylin regulatory peptides: structures,

biological activities mediated by cyclic GMP and pathobiology.

Regul Pept 81(1–3): 25–39.

Forte LR Jr (2004). Uroguanylin and guanylin peptides:

pharmacology and experimental therapeutics. Pharmacol Ther

104: 137–162.

Gariepy J, Judd AK, Schoolnik GK (1987). Importance of

disulfide bridges in the structure and activity of Escherichia coli

enterotoxin ST1b. Proc Natl Acad Sci U S A 84: 8907–8911.

Hamra FK, Forte LR, Eber SL, Pidhorodeckyj NV, Krause WJ,

Freeman RH, et al. , et al. (1993). Uroguanylin: structure and

activity of a second endogenous peptide that stimulates

intestinal guanylate cyclase. Proc Natl Acad Sci USA 90:

10464–10468.

Hamra FK, EberSL Chin DT, Currie MG, Forte LR (1997).

Regulation of intestinal uroguanylin/guanylin receptor-

2017 | Vol. 5 | Iss. 2 | e00295




mediated responses by mucosal acidity. Proc Natl Acad Sci

USA 94: 2705–2710.

Hannig G, Tchernychev B, Kurtz CB, Bryant AP, Currie MG,

Silos-Santiago I (2014). Guanylate cyclase-C/cGMP: an

emerging pathway in the regulation of visceral pain. Front Mol

Neurosci 7: 31.

Harris LA, Crowell MD (2007). Linaclotide, a new direction in

the treatment of irritable bowel syndrome and chronic

constipation. Curr Opin Mol Ther 9: 403–410.

Heidelbaugh JJ, Stelwagon M, Miller SA, Shea EP, Chey WD

(2015). The spectrum of constipation-predominant irritable

bowel syndrome and chronic idiopathic constipation: US

survey assessing symptoms, care seeking, and disease burden.

Am J Gastroenterol 110: 580–587.

Hess B, Kutzner C, van der Spoel D, Lindahl E (2008).

GROMACS 4: algorithms for Highly Efficient, Load-Balanced,

and Scalable Molecular Simulation. J Chem Theory Comput 4:

435–447.

Hughes JM, Murad F, Chang B, Guerrant RL (1978). Role of

cyclic GMP in the action of heat-stable enterotoxin of

Escherichia coli. Nature 271: 755–756.

Kita T, Smith CE, Fok KF, Duffin KL, Moore WM, Karabatsos

PJ, et al. , et al. (1994). Characterization of human

uroguanylin: a member of the guanylin peptide family. Am J

Physiol 266(2 Pt 2): F342–F348.

Klodt J, Kuhn M, Marx UC, Martin S, Rosch P, Forssmann

WG, et al. (1997). Synthesis, biological activity and isomerism

of guanylate cyclase C-activating peptides guanylin and

uroguanylin. J Pept Res 50: 222–230.

Lembo AJ, Schneier HA, Shiff SJ, Kurtz CB, MacDougall JE,

Jia ZD, et al. (2011). Two randomized trials of linaclotide for

chronic constipation. N Engl J Med 365: 527–536.

Marx UC, Klodt J, Meyer M, Gerlach H, Rosch P, Forssmann

WG, et al. (1998). One peptide, two topologies: structure and

interconversion dynamics of human uroguanylin isomers.

J Pept Res 52: 229–240.

Molecular Operating Environment2015, v., Chemical

Computing Group, Montreal, Canada, Avaliable at: http://

www.chemcomp.com.(accessed: 01 September 2016)

Nataro JP, Kaper JB (1998). Diarrheagenic Escherichia coli.

Clin Microbiol Rev 11: 142–201.

Ozaki H, Sato T, Kubota H, Hata Y, Katsube Y, Shimonishi Y

(1991). Molecular structure of the toxin domain of heat-stable

enterotoxin produced by a pathogenic strain of Escherichia

coli. A putative binding site for a binding protein on rat

intestinal epithelial cell membranes. J Biol Chem 266: 5934–

5941.

Pitari GM (2013). Pharmacology and clinical potential of

guanylyl cyclase C agonists in the treatment of ulcerative

colitis. Drug Des Devel Ther 7: 351–360.

Shailubhai K, Yu HH, Karunanandaa K, Wang JY, Eber SL,

Wang Y, et al. (2000). Uroguanylin treatment suppresses polyp

formation in the Apc(Min/+) mouse and induces apoptosis in

human colon adenocarcinoma cells via cyclic GMP. Cancer

Res 60: 5151–5157.

Shailubhai K, Comiskey S, Foss JA, Feng R, Barrow L, Comer

GM, et al. (2013). Plecanatide, an oral guanylate cyclase C

agonist acting locally in the gastrointestinal tract, is safe and

well-tolerated in single doses. Dig Dis Sci 58: 2580–2586.

Shailubhai K, Palejwala V, Arjunan KP, Saykhedkar S, Nefsky

B, Foss JA, et al. (2015). Plecanatide and dolcanatide, novel

guanylate cyclase-C agonists, ameliorate gastrointestinal

inflammation in experimental models of murine colitis. World

J Gastrointest Pharmacol Ther 6: 213–222.

Shimonishi Y, Hidaka Y, Koizumi M, Hane M, Aimoto S,

Takeda T, et al. (1987). Mode of disulfide bond formation

of a heat-stable enterotoxin (STh) produced by a human

strain of enterotoxigenic Escherichia coli. FEBS Lett 215:

165–170.

Skelton NJ, Garcia KC, Goeddel DV, Quan C, Burnier JP

(1994). Determination of the solution structure of the peptide

hormone guanylin: observation of a novel form of topological

stereoisomerism. Biochemistry 33: 13581–13592.

Steinbrecher KA (2014). The multiple roles of guanylate

cyclase C, a heat stable enterotoxin receptor. Curr Opin

Gastroenterol 30: 1–6.

Suares NC, Ford AC (2011). Prevalence of, and risk factors

for, chronic idiopathic constipation in the community:

systematic review and meta-analysis. Am J Gastroenterol 106:

1582–1591.

Synergy Pharmaceuticals Inc. (2015a) Synergy pharmaceuticals

announces positive results in the first phase 3 trial of

plecanatide in patients with chronic idiopathic constipation

(CIC) [Press release]

Synergy Pharmaceuticals Inc. (2015b) Synergy pharmaceuticals

initiates second phase 3 clinical trial of plecanatide in patients

with irritable bowel syndrome with constipation [Press

Release]

Vaandrager AB, Bot AG, De Jonge HR (1997). Guanosine

30,50-cyclic monophosphate-dependent protein kinase II

mediates heat-stable enterotoxin-provoked chloride secretion

in rat intestine. Gastroenterology 112: 437–443.

Whitaker TL, Witte DP, Scott MC, Cohen MB (1997).

Uroguanylin and guanylin: distinct but overlapping patterns of

messenger RNA expression in mouse intestine.

Gastroenterology 113: 1000–1006.

Yoshimura S, Ikemura H, Watanabe H, Aimoto S, Shimonishi

Y, Hara S, et al. (1985). Essential structure for full

enterotoxigenic activity of heat-stable enterotoxin produced

by enterotoxigenic Escherichia coli. FEBS Lett 181: 138–142.


2017 | Vol. 5 | Iss. 2 | e00295


http://www.chemcomp.com.(accessed

http://www.chemcomp.com.(accessed

Supporting Information

Additional Supporting Information may be found online

in the supporting information tab for this article:

Figure S1. The graphs represent the RMSD variation dur-

ing the simulation for each peptide. The RMSD is calcu-

lated taking as reference the structure of the most

representative cluster for each peptide. In the case of Ple-

canatide-pH>5.0 (Asn-/Glu), three major clusters were

present. All the frames included in the interval between 0

and 0.1 �A are part of the representative clusters for each

peptide.

Figure S2. Residues RMS fluctuation. It is clear how the

fluctuation is minimal for STh and Linaclotide due to the

higher rigidity of the two peptide. On the contrary, a

higher flexibility is obtained for the four Plecanatide pep-

tides, according to the different protonation states.

2017 | Vol. 5 | Iss. 2 | e00295




therapeutically targeting guanylate cyclase‐c...

Documents