PRP
`
`ORIGINAL ARTICLE
`
`Open Access
`
`Therapeutically targeting guanylate cyclase-C:
`computational modeling of plecanatide, a uroguanylin
`analog
`Andrea Branca le 1
`2
`Gary S Jacob
`'School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, United Kingdom
`2Synergy Pharmaceuticals, New York, New York
`
`$, Kunwar Shailubhai
`
`2
`
`, Salvatore Feria 1, Antonio Ricci 1, Marcella Bassetto 1 &
`
`Keywords
`guanylate Cyclase (, 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: BrancaleA@cardiff.ac.uk
`
`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%
`
`© 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
`
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`Computational Modeling of Plecanatide, a Uroguanyl in Analog
`
`B. Andrea et al
`
`of the North American population, is chaUenging to treat
`and poses a significant burden on health resources
`(Suares and Ford 2011).
`Guanylate cydase C (GC C) receptors play a crucial
`role in the maintenance of normal bowel function and
`thus have potential as a target fur 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 linadotide have been developed for the
`treatment of two of these disorders, CIC and JBS 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 preferentiaUy 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 cydic 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 fur 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 £ . coli
`enterotoxin (Sfh) 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 aUows uroguanylin and
`guanylin to adopt two topological isofurms (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
`fur 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 JBS C. Linadotide, a synthetic ana
`log of STh, is available for the treatment of CIC and JBS
`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 JBS 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
`
`C E~Y c C N
`' ~
`c~
`P
`A
`cGTC
`V
`Linaclotide
`
`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 sensi ng
`residue (aspartatic acid, D) of uroguanylin is replaced w it h another
`pH sensing resi due (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, com prised of residues 5 17 of the full heat stable
`enterotoxin protein representing the core bioactive pharmacophore of
`pepti de. (A). STh and linaclotide both lack pH sensing residues on
`their N terminal ends and are stabilized into a constit utively active
`conformer by the presence of 3 disuWide bonds. Structurally, the
`pepti des differs by the replacement of leucine (L) in STh w it h tyrosine
`(V-) in linaclotide.
`
`2017 I Vol. 5 I lss. 2 I e00295
`Page 2
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`B. Andrea et al.
`
`Computational Modeling of Plecanatide, a Uroguanylin Analog
`
`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 (PDE ID: lUYA)
`and the crystal structure of STa (PDE ID: lETN)
`(Fig. Sl) 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. Sl ).
`
`© 2017 The Authors Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
`British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics
`
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`Computational Modeling of Plecanatide, a Uroguanylin Analog
`
`B. Andrea er al.
`
`Table 1. Representative cluster analysis.
`
`Compound and
`test condition
`
`Cluster % On the
`total frames2
`size'
`
`Average
`RM SD (A) ± SD3
`
`STh
`Linacl otide
`Plecanatide
`pH>7.0
`Pl eca natide
`pH 5.0 (Asp /Glu)4
`
`Plecanatide
`pH 5.0 (Asp/Glu )
`Pl eca natide pH<2.0
`
`2273
`3592
`1108
`
`524
`497
`446
`610
`
`709
`
`56
`89
`28
`
`13
`12
`11
`15
`
`18
`
`0.1252 ± 0.0821
`0.0715 ± 0.0421
`0.1827 ± 0.0862
`
`0.3208 ± 0.1325
`0.2425 ± 0.1044
`0.1252 ± 0.1107
`0.2885 ± 0.1363
`
`0.2115 ± 0.0825
`
`'Th e cluster size refers to the number of frames that are forming the
`cluster. The total number of frames is 3999.
`2Th e % is calculated on the total number of frames.
`3Th e average RM SD is calculated against the most representative clus
`ter conformation for each peptide.
`"Three rep-esentative clust ers were obtained for this peptide.
`
`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 Sfh show very
`little deviation. The fact that the snapshots show a high
`degree of overlap across all rounds of simulations indi
`cates that Sfh 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, Gius.
`However, unlike Glu3, its side chain is oriented away
`from the interaction loop and, given its position between
`the two disulfide bonds, Gius 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 linadotide which are not affected
`by pH variations (Busby et al. 2010). For these reasons,
`the protonation state of Gius 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
`
`N-tenninus
`
`N-tenninus
`
`■ STh
`linaclotide
`
`Figure 2. MD simulations of STh and linaclotide. (A) Overla pping snapsho1S of STh from MD simulations reveal that the peptide adopts a single
`stable structlJ'e with little flexibility. (BJ Superimposition of represen ta1ive structures from 1he STh and linaclotide. Variations between structures at
`the C terminus reflects changes in conformatio n indu::ed by 1he additional tyrosine of linadotide. Simulations reveal that linadotide adopts a
`similar conformation as STh especially so w ithin the reg ion of the GC C interaction loop (•). MD, M olecular dynamics.
`
`2017 I Vol. 5 I lss. 2 I e00295
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`Computational Modeling of Plecanatide, a Uroguanylin Ana log
`
`B. Andrea et al.
`
`(A)
`N-tcrminus
`
`C-terminus
`
`(D)
`
`(B)
`
`*
`
`■ STh ■ PICCllllatidc
`
`Figure 3. Overlapping snapshots of STh and plecanande at pH values 5.0 (A D). 2.0 (E) and >7.0 (F) using MD simulations. (A-C) Overlay of the
`A: 2.38 A; B:2.48 A; C:3 44 A) (D) The
`three predominant Asp /Glu conforma tions of plecanatide and STh at pH 5.0 (RMSD values
`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 th ose of STh with no CNerlap of interaction loop (RMSD value 3.45 A). (F) Simulations of the double negative form of
`plecanande. As p/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
`
`protonation configurations were analyzed. In one, Asp2 is
`protonated (Asp/Glu ), whereas in the other, Glu3 is pro
`tonated (Asp /Glu). These ionii.ation 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 confonnations
`of plecanatide and STh, and Figure 3D shows the pre
`dominant Asp/Glu confonnation 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 confonnations 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. 3£) . 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 ionii.ation state of pleca
`natide at pH 5, an interaction occurred between the
`negatively charged acidic side chain of Glu3 residue in
`the N tenninus 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
`
`© 2017 The Authors Pharmacology Research & Perspectives pubished by John Wiey & Sons Ltd,
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`Computational Modeling of Plecanatide, a Uroguanylin Analog
`
`B. Andrea et al
`
`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 ionii.ation 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 dusters. With an RMSD of 1.28 A, the structure
`of linadotide 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
`
`Table 2. RMSD com parison between STh and the most representa
`tive clusters of the differen t peptides sim ulated.
`
`Compound and Test Condition
`
`RMSD-A
`
`Linaclotide
`Uroguanylin
`Plecanatide pH>7 o
`Plecanatide pH 5 0 (Asp-/GI u)
`Plecanatide pH 5 0 (Asp/GI u )
`Plecanatide pH<2 o
`
`1.28
`1.2 2.34 1
`2 .54
`3.44, 2.48, 2.382
`1.93
`3.45
`
`' Range of RMSD values between STh and the 10 NMR conformations
`of uroguanyli n.
`2Three representative cl usters (>10%
`see methods section) were
`obtained for this pepti de. RMSD comparisons of the different peptides
`analyzed were used to quantify the similarit ies between the struc
`lures. Lower values indicate higher structural similarit y with STh refer
`ence mdecule.
`
`the AsJ)'Glu
`in
`Figure 4. Structure of plecanatide at pH 5.0,
`ionization state. Interaction (•) between the negative charge of Glu3
`resi due on the pH sens~ive N terminus an d Asn9 residue in the
`interaction loop potentially serves to stabilize this active conformation
`of plecanatide.
`
`N-tenninus
`
`C-tenninus
`
`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
`substit ution Asp3/Glu3 is present.
`
`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 dosely mimics plecanatide. The
`
`2017 I Vo l. 5 I lss. 2 I e00295
`Page 6
`
`© 20 17 The Authors Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
`British Pharmacologi<.al Society and Ameri<.an Society for Pharmacology and Experimental Therapeutics
`
`Bausch Health Ireland Exhibit 2019, Page 6 of 10
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`
`

`

`B Andrea et al.
`
`Computational Modeling of Plecanatide, a Uroguanylin Analog
`
`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.
`
`D i scussion
`
`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
`mucosa! 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. O