Therapeutically Targeting Guanylate

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ORIGINAL ARTICLE Therapeutically targeting guanylate cyclase-C: computational modeling of plecanatide, a uroguanylin analog Andrea Brancale1 , Kunwar Shailubhai2, Salvatore Ferla1, Antonio Ricci1, Marcella Bassetto1 & Gary S Jacob2 1School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, United Kingdom 2Synergy Pharmaceuticals, New York, New York

Keywords Abstract guanylate Cyclase-C, linaclotide, molecular dynamics, plecanatide, uroguanylin Plecanatide is a recently developed guanylate cyclase-C (GC-C) agonist and the first uroguanylin analog designed to treat chronic idiopathic constipation (CIC) Correspondence and irritable bowel syndrome with constipation (IBS-C). GC-C receptors are Andrea Brancale, Cardiff School of Pharmacy found across the length of the intestines and are thought to play a key role in and Pharmaceutical Sciences, Cardiff fluid regulation and electrolyte balance. Ligands of the GC-C receptor include University, Redwood Building, King Edward endogenous agonists, uroguanylin and guanylin, as well as diarrheagenic, VII Avenue, Cardiff, CF10 3NB. Escherichia coli heat-stable enterotoxins (ST). Plecanatide mimics uroguanylin Tel: +44 (0)29 2087 4485; Fax: +44(29) 2087 4149; in its 2 disulfide-bond structure and in its ability to activate GC-Cs in a pH- E-mail: [email protected] dependent manner, a feature associated with the presence of acid-sensing residues (Asp2 and Glu3). Linaclotide, a synthetic analog of STh (a 19 amino acid Funding Information member of ST family), contains the enterotoxin’s key structural elements, Financial Support for this study provided by including the presence of three disulfide bonds. Linaclotide, like STh, activates Synergy Pharmaceuticals Inc. We also GC-Cs in a pH-independent manner due to the absence of pH-sensing residues. acknowledge the support of the Life Science In this study, molecular dynamics simulations compared the stability of pleca- Research Network Wales grant # NRNPGSep14008, an initiative funded natide and linaclotide to STh. Three-dimensional structures of plecanatide at through the Welsh Government’s Ser Cymru various protonation states (pH 2.0, 5.0, and 7.0) were simulated with GRO- program. MACS software. Deviations from ideal binding conformations were quantified using root mean square deviation values. Simulations of linaclotide revealed a Received: 15 August 2016; Revised: 23 rigid conformer most similar to STh. Plecanatide simulations retained the flexi- November 2016; Accepted: 30 November ble, pH-dependent structure of uroguanylin. The most active conformers of ple- 2016 canatide were found at pH 5.0, which is the pH found in the proximal small Pharma Res Per, 5(2), 2017, e00295, intestine. GC-C receptor activation in this region would stimulate intraluminal doi: 10.1002/prp2.295 fluid secretion, potentially relieving symptoms associated with CIC and IBS-C. doi: 10.1002/prp2.295 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 tract, creating a burden on healthcare resources and leading to signiﬁcant negative impact on quality of life (Hei- Chronic idiopathic constipation (CIC) and irritable bowel delbaugh et al. 2015). These disorders are characterized syndrome with constipation (IBS-C) are two of the most by diminished stool frequency, straining and abdominal common conditions affecting the gastrointestinal (GI) pain (IBS-C) or discomfort (CIC). CIC alone affects 14%

ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, 2017 | Vol. 5 | Iss. 2 | e00295 British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics. Page 1 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. Computational Modeling of Plecanatide, a Uroguanylin Analog B. Andrea et al. of the North American population, is challenging to treat acid residues on its N-terminus (Hamra et al. 1997). The and poses a significant burden on health resources absence of these residues within STh allows it to bypass pH (Suares and Ford 2011). checkpoints governing GC-C receptor activation, allowing Guanylate cyclase-C (GC-C) receptors play a crucial for supraphysiological activation of GC-C along the length role in the maintenance of normal bowel function and of the small intestine and colon (Hamra et al. 1997). thus have potential as a target for pharmaceutical inter- Pharmacologic agonists that mimic the activity of vention in to treat numerous functional gastrointestinal known GC-C agonists have been developed to treat disorders. The GC-C receptor is a membrane-spanning patients with CIC and IBS-C. Linaclotide, a synthetic ana- protein uniformly expressed along the epithelial brush log of STh, is available for the treatment of CIC and IBS- border throughout the intestine (Forte 1999). Recently C (Lembo et al. 2011). Like STh, linaclotide has three plecanatide and linaclotide have been developed for the disulfide bonds and demonstrates pH-independent activa- treatment of two of these disorders, CIC and IBS-C tion of GC-C receptors (Fig. 1) (Busby et al. 2010). (Shailubhai et al. 2013). Plecanatide is a recently developed GC-C agonist and The GC-C receptor is activated by its endogenous pep- uroguanylin analog that is currently in Phase 3 trials for tides uroguanylin and guanylin that differentially bind to CIC and IBS-C (Synergy Pharmaceuticals Inc 2015a,b). the receptor in the varying pH environments found along Plecanatide is an orally administered, pH-dependent ago- the GI tract (Fan et al. 1997). Uroguanylin is primarily nist of the GC-C receptor that shares the structural and expressed and preferentially binds GC-C receptors in the physiological characteristics of uroguanylin. Plecanatide slightly acidic (pH 5-6) regions of the duodenum and has two disulfide bonds, similar to uroguanylin, and con- jejunum (Forte 1999). Guanylin is primarily expressed in tains two acidic N-terminal amino acids allowing the two the ileum and colon, activating GC-C receptors under molecules to maintain the same pH-dependent binding more basic conditions (pH 7 -8) (Kita et al. 1994). Bind- ing of uroguanylin or guanylin to the GC-C receptor initiates 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 electrolytes and water into the intestinal lumen resulting in diarrhea (Brierley 2012). Figure 1. Amino acid structures of guanylate cyclase-C receptor Studies have shown STh to be 10 times more potent than agonists examined in this study. Synthetic analogs linaclotide and uroguanylin and 100 times more potent than guanylin in plecanatide share similar amino acid sequences with GC-C agonists binding to GC-C receptors (Hamra et al. 1993). The X-ray STh and uroguanylin, respectively. Plecanatide, like uroguanylin structure of STh reveals that the molecule is locked into a contains two pH-sensing residues on its N-terminus. The pH-sensing residue (aspartatic acid, D) of uroguanylin is replaced with another constitutively active, right-handed spiral formation stabi- pH-sensing residue (glutamic acid, E) in plecanatide (green). In this lized by three intrachain disulfide bridges in a 1-4/2-5/3-6 study, the pH-sensing aspartic acid and glutamic acid residues of pattern (Gariepy et al. 1987; Ozaki et al. 1991; Shimonishi plecanatide were differentially protonated to reflect pH values 2.0 et al. 1987). Unlike STh, the endogenous peptides have only (Asp2, Glu3), 5.0 (Asp2-,Glu3; Asp2, Glu3-) & ≥7.0 (Asp2-, Glu3-). two disulfide bridges which likely results in their improved Simulations of the crystal structure of STh used a truncated version of – flexibility; Klodt et al. (1997). The flexibility afforded by the full toxin, comprised of residues 5 17 of the full heat-stable enterotoxin protein representing the core bioactive pharmacophore of absence of a third disulfide bridge allows uroguanylin and peptide. (A). STh and linaclotide both lack pH-sensing residues on guanylin to adopt two topological isoforms (A and B) of their N-terminal ends and are stabilized into a constitutively active which only the A form is biologically active (Marx et al. conformer by the presence of 3 disulfide bonds. Structurally, the 1998; Skelton et al. 1994). The pH-dependent activity of peptides differs by the replacement of leucine (L) in STh with tyrosine uroguanylin is linked to two charged acid-sensing aspartic (Y*) in linaclotide.

2017 | Vol. 5 | Iss. 2 | e00295 ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, Page 2 British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics. B. Andrea et al. Computational Modeling of Plecanatide, a Uroguanylin Analog characteristics (Fig. 1) (Shailubhai et al. 2013). Given the considered to be the pharmacophore of the molecule experimental challenges of studying the unique character- required for maximum biological activity (Ozaki et al. istics of its pH-sensitive structure, computational methods 1991; Yoshimura et al. 1985). were employed to characterize behavior of plecanatide at Four protonation states of plecanatide were modeled various pH states. by placing the appropriate charge on Asp2 and Glu3, Molecular dynamics (MD) simulations compared the reflecting the most abundant species at three pH environ- flexibility and conformation of plecanatide and linaclotide. ments: pH 2.0 (Asp2, Glu3), pH 5.0 (Asp2-, Glu3; Asp2, As expected, linaclotide was shown to adopt rigid confor- Glu3-), and pH 7.0 (Asp2-, Glu3-) (Fig. 1 circle). The ini- mations, which do not deviate significantly from the struc- tial structure of each peptide was placed in a cubic box ture of STh. In contrast, plecanatide showed greater with TIP 3P water and energy minimized using a Steepest flexibility and an ability to adopt several conformations Descent Minimization Algorithm. The system was equili- which vary in response to pH values (2.0, 5.0 and 7.0). brated via a 50 ps MD simulation at 310 K in a NVT Active plecanatide conformations were more similar to canonical environment followed by an additional 50 ps uroguanylin than to the STh peptide, suggesting that pleca- simulation at constant pressure of 1 atm (NPT). After the natide has a similar pH-dependent activity profile as uro- equilibration phases, a 500 ns MD simulations were per- guanylin and that plecanatide’s activity can be differentially formed at constant temperature (310 K) and pressure regulated in the GI tract, with higher activity in the more with a time step of 2 fs. The system energy and peptide acidic proximal small intestine and lower activity in the spatial coordinates (trajectory file) were stored every more basic distal small intestine and colon. 300 ps for further studies. All MD simulations were run in triplicate for each peptide. Materials and Methods After removing the first 100 ns, considered as system stabilization time, the remaining 400 ns of the MD trajectory of every single run for each group of triplicate exper- Peptide preparation iments were combined using the tricat function. The The NMR structures of uroguanylin (PDB ID: 1UYA) combined trajectories (3999 frames) were examined using and the crystal structure of STa (PDB ID: 1ETN) the g-cluster function, setting gromos as the clustering (Fig. S1) were used as starting points for the MD simula- method with an RMSD cut-off of 0.1 nm. The different tions (Ozaki et al. 1991). The STa family of heat stable structural cluster groups were obtained as a pdb file. peptides includes STh and STp (Nataro and Kaper 1998). Cluster groups representing at least 10% of the total pop- The series of experiments used in this study used the STh ulation for each peptide were selected as the most repre- sequence as reference. sentative structure for that peptide. Structural models of plecanatide, linaclotide, and STh were built by appropriately modifying the uroguanylin and RMSD comparisons STa sequences, respectively, using the builder tools in molecular operating environment [(MOE 2015.4) www.chemc The representative cluster conformations of the different omp.com]. (Molecular Operating Environment 2015). peptides were used for the RMSD comparison against the Simulations and root mean standard deviation (RMSD) main conformation of the STh cluster. values of linaclotide and each configuration of plecanatide RMSD comparisons were performed using MOE 2015.4 were compared to the enteric pathogen, STh, which was with the major STh cluster structure serving as a reference selected as the reference peptide because of its enhanced for the superimposition of other peptide conformations. affinity for the GC-C receptor compared to other agonists. The structural rigidity of STh confers one main Results conformation that would bind the receptor. Similarities to this one conformer would reflect a drugs ability to MD clustering activate GC-C. Table 1 shows the results obtained from the structural cluster calculations. From this data it is possible to appre- MD simulations ciate how flexible plecanatide is, compared to STh and All MD simulations of plecanatide, STh, and linaclotide Linaclotide. The latter peptides generated more populated were performed and analyzed using the GROMACS 4.5 cluster than any of the plecanatide forms. RMSD analysis simulation package (Hess et al. 2008). MD simulations of of each MD simulations calculated against the most rep- the structure of STh used residues 6–18 of the full heat- resentative clusters also confirm this observation stable enterotoxin protein (C-C-E-L-C-C-N-P-A-C-T-G-C), (Fig. S1).

Table 1. Representative cluster analysis. substitution and an additional residue at the C-terminus,

Compound and Cluster % On the Average also reveal a rigid molecule that adopts a similar confor- test condition size1 total frames2 RMSD (A) SD3 mation as STh (Fig. 2B). The rigidity of this peptide was also confirmed by a RSM fluctuation analysis of the MD STh 2273 56 0.1252 0.0821 trajectory (Supplemental Information, Fig. S2). Some Linaclotide 3592 89 0.0715 0.0421 variability can be seen within the C-termini of the pep- Plecanatide – 1108 28 0.1827 0.0862 pH>7.0 tides, likely due to the extra amino acid in linaclotide Plecanatide – 524 13 0.3208 0.1325 compared to STh. Moreover, the conformation of the pH 5.0 (Asp-/Glu)4 497 12 0.2425 0.1044 interaction loops (regions binding the GC-C receptor) is 446 11 0.1252 0.1107 homologous between the two molecules (Fig. 2B *) Plecanatide – 610 15 0.2885 0.1363 (Ozaki et al. 1991). pH 5.0 (Asp/Glu-) Plecanatide – pH<2.0 709 18 0.2115 0.0825 MD simulations of plecanatide 1The cluster size refers to the number of frames that are forming the cluster. The total number of frames is 3999. MD simulations of plecanatide were conducted by alter- 2The % is calculated on the total number of frames. ing the amino acid sequence of the NMR structure of 3 The average RMSD is calculated against the most representative clus- uroguanylin (Marx et al. 1998). Because pH has been ter conformation for each peptide. shown to alter the ability of uroguanylin to activate GC-C 4Three representative clusters were obtained for this peptide. receptors, simulations were conducted on the four ionization states of plecanatide’s structure, reflecting three dif- MD simulations of STh and linaclotide ferent pH values, by altering the protonation states of In addition to providing information on structure, the Asp2 and Glu3 residues. It should be noted that pleca- simulations also provided information on the degree of natide contains an additional pH-sensitive residue, Glu5. internal rigidity and flexibility within the peptide. This However, unlike Glu3, its side chain is oriented away can be observed in the MD simulations shown in Fig- from the interaction loop and, given its position between ure 2A, in which overlapping snapshots of STh show very the two disulfide bonds, Glu5 does not have the confor- little deviation. The fact that the snapshots show a high mational freedom to affect the orientation of the loop degree of overlap across all rounds of simulations indi- itself. Furthermore, this specific residue is highly con- cates that STh is a fairly rigid molecule with little internal served across the whole range of GC-C binding peptides, flexibility. This constrained geometry is thought to be and includes STh and linaclotide which are not affected established by the three disulfide bonds of the peptide by pH variations (Busby et al. 2010). For these reasons, leading to a structural rigidity and high binding affinity the protonation state of Glu5 should not affect the activ- of STh for the GC-C receptor (Ozaki et al. 1991). ity of uroguanylin and plecanatide. MD simulations of linaclotide, which differs from the To represent plecanatide at pH 5.0, which corresponds STh peptide used in the simulations by one amino acid to the pH of the duodenum and proximal jejunum, two

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 ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, Page 4 British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics. B. Andrea et al. Computational Modeling of Plecanatide, a Uroguanylin Analog

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. protonation configurations were analyzed. In one, Asp2 is and dynamics of the peptide at pH 2.0. The plecanatide protonated (Asp/Glu-), whereas in the other, Glu3 is pro- conformations observed at this pH differ from those of tonated (Asp-/Glu). These ionization states were based on STh (Fig. 3E). Based on these results, plecanatide is unli- the consideration that, as these residues are on the flexible kely to adopt a conformation capable of binding to GC-C N-terminus and exposed to solvent, their pKa values at this highly acidic pH level, a feature which would would be between 3.5 and 4.5 (values dependent on the mimic uroguanylin’s inability to activate GC-C receptors input peptide conformation as calculated on http://bio at this pH. physics.cs.vt.edu/H++, version 3.2) (Anandakrishnan et al. Simulations of the double negative form of plecanatide 2012); hence, the monoprotonated states would likely be (Asp-/Glu-), which represent the protonation state present at pH 5.0. Simulations of the two protonation observed at pH > 7.0, reveal a single predominant pleca- states indicate that plecanatide is flexible at this pH and natide structure (Fig. 3F). The portion of the peptide that can adopt several conformations. Figures 3A–C show an interacts with the GC-C receptor adopts a different con- overlay of the three predominant Asp-/Glu conformations formation in plecanatide than in STh indicating dimin- of plecanatide and STh, and Figure 3D shows the pre- ished activity of the molecule at this pH value. dominant Asp/Glu- conformation of plecanatide and STh. Interestingly, in the Asp/Glu- ionization state of pleca- In two of these structures (Fig. 3A and D), the interaction natide at pH 5, an interaction occurred between the loop (*) of plecanatide overlays well with that of STh, negatively charged acidic side chain of Glu3 residue in indicating that these two conformations of plecanatide the N-terminus and the positively charged side chain of are capable of binding to and subsequently activating GC- Asn9 in the interaction loop (Fig. 4). This interaction C receptors. between the Glu3 residue of the N-terminus and the Asn9 Simulations of the double protonated form of pleca- residue of the interaction loop seems to stabilize plecanatide (Asp/Glu) were conducted to assess the structure natide in its most active conformation at pH 5. This

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 plecanatide were found with the two ionization states representative of pH 5.0 as revealed by the similarity of their interaction loops with the corresponding regions in uroguanylin 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 Figure 4. Structure of plecanatide at pH 5.0, in the Asp/Glu- uroguanylin (Shailubhai et al. 2013). 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 Comparison of MD simulations of plecanatide. RMSD comparisons of the different peptides analyzed were used to quantify the similarities between the structures. 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 representative 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 flexible the peptide is. Interestingly, plecanatide presents some conformations that are very close to the reference STh (RMSD <2 A), suggesting a peak activity for plecanatide at this pH. Additionally, RMSD calculations reveal

Table 2. RMSD comparison between STh and the most representative clusters of the different peptides simulated. Figure 5. Overlay of the most active conformation of plecanatide, Asp/Glu-, (pH 5.0), with the active “A” NMR structure of uroguanylin. Compound and Test Condition RMSD–A The interaction loop of the two peptides overlaps generally very well (*). Some variability is observed in the N-terminus, where the Linaclotide 1.28 – 1 substitution Asp3/Glu3 is present. Uroguanylin 1.2 2.34 Plecanatide - pH>7.0 2.54 Plecanatide – pH 5.0 (Asp-/Glu) 3.44, 2.48, 2.382 interaction was not observed at other pH values nor is it Plecanatide – pH 5.0 (Asp/Glu-) 1.93 expected to occur with uroguanylin as the Asp3 amino Plecanatide – pH<2.0 3.45 acid in uroguanylin would not be of sufﬁcient length to 1Range of RMSD values between STh and the 10 NMR conformations interact with the Asn9 residue in its interaction loop. of uroguanylin. Overall, the highly ﬂexible behavior of plecanatide is 2Three representative clusters (>10% – see methods section) were very similar to the one observed with the parent peptide obtained for this peptide. RMSD comparisons of the different peptides uroguanylin (Fig. 5). Indeed, the NMR structures avail- analyzed were used to quantify the similarities between the struc- able for the latter peptide show a high degree of confor- tures. Lower values indicate higher structural similarity with STh refer- mational variability that closely mimics plecanatide. The ence molecule.

2017 | Vol. 5 | Iss. 2 | e00295 ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, Page 6 British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics. B. Andrea et al. Computational Modeling of Plecanatide, a Uroguanylin Analog that these four variants of plecanatide are the closest that the addition of a third intramolecular bond makes structurally to the active A-form of uroguanylin. This is both STh and linaclotide insensitive to MD perturbations consistent with the data described above, in which the (Ozaki et al. 1991). The structural similarity of these two negative charge of Glu3 interacts with Asn9 in the inter- molecules is reflected by the low RMSD values of 1.28 A action loop, potentially promoting an active conformation for linaclotide. The amino acid substitutions that differen- of plecanatide. These results indicate that, in a manner tiate linaclotide from STh further enhance the pharma- similar to uroguanylin, the activity of plecanatide is not cokinetic stability and proteolytic resistance of linaclotide, “all or nothing” but tunable based upon the pH of the allowing it to remain active across a longer portion of the environment. small intestine (Bharucha and Waldman 2010; Harris and Crowell 2007). The absence of pH-sensing amino acid Discussion residues would additionally give these molecules maximum biological activity across the range of pH environ- Transmembrane GC-C receptors play a critical role in the ments in the GI tract. This lack of focused areas of regulation of fluid and electrolyte homeostasis within the activity may induce excessive fluid secretion and explain GI tract (Brierley 2012; Steinbrecher 2014). This balance the increased incidence of diarrhea associated with lina- is normally maintained by the pH-dependent activation clotide (Busby and Ortiz 2014; Carpick and Gariepy 1993; of these receptors by the endogenous ligands, uroguany- Lembo et al. 2011). lin, and guanylin (Forte 2004). Studies have shown that Plecanatide, a GC-C agonist under investigation for these pH environments are maintained at differential val- IBS-C and CIC, differs from linaclotide in its amino acid ues across the length of the gastrointestinal tract (Daniel sequence and number of disulfide bonds. Plecanatide et al. 1985; Whitaker et al. 1997). In the slightly acidic maintains many of the structural and functional charac- mucosal environments of the duodenum and proximal teristics of endogenous ligand uroguanylin, including the jejunum (pH 5.0-6.0), uroguanylin is 10 times more two disulfide bonds and the two N-terminal pH-sensing potent than in the slightly basic (pH 7.0-8.0) environ- acidic residues, which result in a molecule that can exhi- ments of the ileum and colon (Hamra et al. 1997). In bit differential levels of activity based upon the pH of the contrast, guanylin is significantly more potent at binding environment. As a result, plecanatide has a more targeted GC-C receptors in the ileum and colon (pH 7.0-8.0) than zone of activation, stimulating GC-C receptors under in the duodenum and proximal jejunum (pH 5.0-6.0), acidic conditions in a way that is similar to uroguanylin where it is essentially inactive (Hamra et al. 1997). Struc- (Shailubhai et al. 2015). tural shifts induced by pH environments alter the potency MD simulations of plecanatide in this study revealed of these ligands, allowing them to cooperatively regulate plecanatide to be a flexible structure that is unlike STh or GC-C receptor activation (Hamra et al. 1997). Any per- linaclotide and one that is able to adopt numerous con- turbation in the pH-dependent control of GC-C activa- formations in response to a range of simulated pH envi- tion would disrupt the delicate balance of electrolytes and ronments. Optimally active conformations were observed fluids within the intestines. This disruption could be asso- in pH 5.0 simulations which approximates the pH values ciated with disorders such as obstruction, secretory diar- of the duodenum and proximal jejunum. This pattern of rhea and inflammatory bowel disease (Field 2003; optimal activity is similar to that of uroguanylin, indicat- Fiskerstrand et al. 2012). ing that plecanatide and uroguanylin are similar in both Based on their physiology, GC-C receptors are a structure and function (Shailubhai et al. 2013). In con- promising therapeutic target in the treatment of numer- trast, at simulated pH values of 2.0 and 7.0, plecanatide ous gastrointestinal disorders such as CIC and IBS-C. adopted conformations that would make it less likely to Both are common complaints among all age groups and bind GC-C receptors. Across the range of assessed pH are associated with a substantial burden on healthcare values, MD simulations and RMSD analysis reveal that resources in the US (Drossman et al. 2009). While the the structure of plecanatide and the NMR structure of exact pathologic mechanism of these diseases remains an uroguanylin are similar to each other (Fig. 5), and quite area of intense study, the symptoms (i.e. diminished stool dissimilar to the STh structure (1.93–3.45 A and 1.20– frequency, hard/lumpy stools, straining abdominal symp- 2.34 A vs. STh, respectively). toms) indicate an imbalance in fluid and electrolyte As mentioned previously, the sole difference between homeostasis in the GI tract (Steinbrecher 2014). the structures of uroguanylin and plecanatide is the As an analog of the pathological GC-C agonist STh, replacement of an aspartic acid residue (Asp3) with a glu- linaclotide maintains many structural features of STh, tamic acid residue (Glu3) in plecanatide (Fig. 1). Previous including the presence of three disulfide bonds and an studies have shown that while plecanatide has a similar insensitivity to pH. MD simulations in this study show pH-dependent affinity for the GC-C receptor, it binds

ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, 2017 | Vol. 5 | Iss. 2 | e00295 British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics. Page 7 Computational Modeling of Plecanatide, a Uroguanylin Analog B. Andrea et al. with superior potency than uroguanylin (Shailubhai et al. Bharucha AE, Waldman SA (2010). Taking a lesson from 2000, 2015). Computational models in this study revealed microbial diarrheagenesis in the management of chronic that at pH 5.0, the charged end of the Glu3 residue inter- constipation. Gastroenterology 138: 813–817. acts with the Asn9 residue, potentially serving to stabilize Brierley SM (2012). Guanylate cyclase-C receptor activation: the structure when it forms its most active conformation. unexpected biology. Curr Opin Pharmacol 12: 632–640. Interestingly, the corresponding interaction between Asp3 Busby RW, Ortiz S (2014). Clarification of linaclotide and Asn9 is not present in uroguanylin, probably because pharmacology presented in a recent clinical study of the side chain of Asp3 is not long enough to reach Asn9. plecanatide. Dig Dis Sci 59: 1066–1067. Stabilizing the molecule in this way allows plecanatide to Busby RW, Bryant AP, Bartolini WP, Cordero EA, Hannig G, remain in its active conformer for a longer period of time Kessler MM, et al. (2010). Linaclotide, through activation of at this pH and explain the superior binding profile of ple- guanylate cyclase C, acts locally in the gastrointestinal tract to canatide versus uroguanylin witnessed in earlier studies. elicit enhanced intestinal secretion and transit. Eur J Recognizing the acidic-to-basic pH gradient nature of the Pharmacol 649(1–3): 328–335. GI tract, the simulations in this study suggest that, similar to uroguanylin, plecanatide would be optimally active in Carpick BW, Gariepy J (1993). The Escherichia coli heat-stable the more acidic environment that is present in the proximal enterotoxin is a long-lived superagonist of guanylin. Infect – small intestine of the GI tract. The ability of plecanatide to Immun 61: 4710 4715. activate GC-C receptors in a targeted manner similar to the Daniel H, Neugebauer B, Kratz A, Rehner G (1985). endogenous ligand uroguanylin may help normalize bowel Localization of acid microclimate along intestinal villi of rat movements and explain the low levels of adverse events jejunum. Am J Physiol 248(3 Pt 1): G293–G298. seen with the drug in early clinical trials (Shailubhai et al. Drossman DA, Chey WD, Johanson JF, Fass R, Scott C, Panas 2013). Understanding the structural mechanisms by which R, et al. (2009). Clinical trial: lubiprostone in patients with plecanatide and linaclotide activate GC-C receptors and constipation-associated irritable bowel syndrome–results of respond to pH could shed light on clinical differences two randomized, placebo-controlled studies. Aliment between the two therapeutic options. Pharmacol Ther 29: 329–341. Fan X, Hamra FK, London RM, Eber SL, Krause WJ, Freeman Acknowledgements RH, et al. (1997). Structure and activity of uroguanylin and guanylin from the intestine and urine of rats. Am J Physiol We wish to acknowledge Medical Dynamics for assistance 273(5 Pt 1): E957–E964. in the preparation of this manuscript. We also acknowl- Field M (2003). Intestinal ion transport and the edge the support of the Life Science Research Network pathophysiology of diarrhea. J Clin Invest 111: 931–943. Wales grant no. NRNPGSep14008, an initiative funded through the Welsh Government’s Ser Cymru program. 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 Authorship Contributions 366: 1586–1595. Brancale and Shailubhai participated in research design. Forte LR (1999). Guanylin regulatory peptides: structures, Ferla, Ricci, and Bassetto conducted the experiments. biological activities mediated by cyclic GMP and pathobiology. – – Brancale and Ferla performed data analysis. Brancale, Regul Pept 81(1 3): 25 39. Shailubhai, and Jacob wrote or contributed to the writing Forte LR Jr (2004). Uroguanylin and guanylin peptides: of the manuscript. pharmacology and experimental therapeutics. Pharmacol Ther 104: 137–162. Disclosures Gariepy J, Judd AK, Schoolnik GK (1987). Importance of disulfide bridges in the structure and activity of Escherichia coli K Shailubhai and G S Jacob are employees and stockhold- enterotoxin ST1b. Proc Natl Acad Sci U S A 84: 8907–8911. ers of Synergy Pharmaceuticals. Hamra FK, Forte LR, Eber SL, Pidhorodeckyj NV, Krause WJ, Freeman RH, et al. , et al. (1993). Uroguanylin: structure and References activity of a second endogenous peptide that stimulates Anandakrishnan R, Aguilar B, Onufriev AV (2012). H++ 3.0: intestinal guanylate cyclase. Proc Natl Acad Sci USA 90: – automating pK prediction and the preparation of bimolecular 10464 10468. structures for atomistic molecular modeling and simulation. Hamra FK, EberSL Chin DT, Currie MG, Forte LR (1997). Nucleic Acids Res 40: W573–541. Regulation of intestinal uroguanylin/guanylin receptor-

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Supporting Information present. All the frames included in the interval between 0 and 0.1 A are part of the representative clusters for each Additional Supporting Information may be found online peptide. in the supporting information tab for this article: Figure S2. Residues RMS fluctuation. It is clear how the fluctuation is minimal for STh and Linaclotide due to the Figure S1. The graphs represent the RMSD variation dur- higher rigidity of the two peptide. On the contrary, a ing the simulation for each peptide. The RMSD is calcu- higher flexibility is obtained for the four Plecanatide pep- lated taking as reference the structure of the most tides, according to the different protonation states. representative cluster for each peptide. In the case of Ple- canatide-pH>5.0 (Asn-/Glu), three major clusters were

2017 | Vol. 5 | Iss. 2 | e00295 ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, Page 10 British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.