Chemical synthesis and structural analysis of guanylate cyclase C agonist linaclotide

Article abstract of DOI:10.1016/j.cclet.2018.01.005

Guanylate cyclase C (GC-C) is an important receptor protein expressed by intestinal epithelial cells, and its dysregulation leads to severe intestinal diseases. Linaclotide is a 14-amino acid peptide approved by the FDA for the treatment of irritable bowel syndrome with constipation (IBS-C), which activates guanylate cyclase C to accelerate intestinal transit. Drug molecule design based on structural information plays a crucial role and the activity of linaclotide still need to improve, while the structure of linaclotide remains unknown. In this work, linaclotide and its D-enantiomer were obtained through Fmoc solid phase peptide synthesis method and co-crystalized through racemic crystallization. The crystal structure showed that linaclotide has a tight, three-beta turns structure immobilized by three pairs of disulfide bonds.

Full text of DOI:10.1016/j.cclet.2018.01.005

Accepted Manuscript
Title: Chemical synthesis and structural analysis of guanylate
cyclase C agonist linaclotide
Authors: Chenchen Chen, Shuai Gao, Qian Qu, Pengcheng
Mi, Anjin Tao, Yi-Ming Li
PII:
DOI:
Reference:
S1001-8417(18)30005-6
https://doi.org/10.1016/j.cclet.2018.01.005
CCLET 4395
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Chinese Chemical Letters
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Accepted date:
20-11-2017
8-12-2017
4-1-2018
Please cite this article as: Chenchen Chen, Shuai Gao, Qian Qu,
Pengcheng Mi, Anjin Tao, Yi-Ming Li, Chemical synthesis and structural
analysis of guanylate cyclase
C
agonist linaclotide, Chinese Chemical
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Communication
Chemical synthesis and structural analysis of guanylate cyclase C agonist linaclotide
Chenchen Chena,b, Shuai Gaoc, Qian Quc, Pengcheng Mid, Anjin Taod, Yi-Ming Lic,

a High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei 230031, China
b School of Life Sciences, University of Science and Technology of China, Hefei 230026, China
c School of Biological and Medical Engineering, Hefei University of Technology, Hefei 230009, China
d Hybio Pharmaceutical Co., Ltd., Shenzhen 518057, China
 Corresponding author.
E-mail address: ymli@hfut.edu.cn
Graphical Abstract:
Linaclotide and its D-enantiomer were obtained through Fmoc solid phase peptide synthesis method and co-crystalized through
racemic crystallization. The crystal structure showed that linaclotide has a tight, three-beta turns structure immobilized by three
pairs of disulfide bonds.
ARTICLE INFO
ABSTRACT
Article history:
Guanylate cyclase C (GC-C) is an important receptor protein expressed by intestinal epithelial
cells, and its dysregulation leads to severe intestinal diseases. Linaclotide is a 14-amino acid
peptide approved by the FDA for the treatment of irritable bowel syndrome with constipation
(IBS-C), which activates guanylate cyclase C to accelerate intestinal transit. Drug molecule
design based on structural information plays a crucial role and the activity of linaclotide still
need to improve, while the structure of linaclotide remains unknown. In this work, linaclotide
and its D-enantiomer were obtained through Fmoc solid phase peptide synthesis method and co-
crystalized through racemic crystallization. The crystal structure showed that linaclotide has a
tight, three-beta turns structure immobilized by three pairs of disulfide bonds.
Received 20 December 2017
Received in revised form 11 December 2017
Accepted 18 December 2017
Available online
Keywords:
Linaclotide
Guanylyl cyclase
Protein chemical synthesis
Fmoc solid phase peptide synthesis
Racemic Crystallization
Irritable bowel syndrome (IBS) is a common chronic functional gastrointestinal disorder and studies show that about 7%-15% of
North Americans and about 11.5% of Europeans are affected [1-4]. IBS patients have frequent and intermittent abdominal pain and
discomfort, which not only adversely affect the patient’s health, but also reduce work efficiency and increase the use of related medical
resources [5,6]. IBS with constipation (IBS-C) patients account for about one-third of the total number of IBS patients, and the number
of female patients is more than male [7]. Apart from abdominal pain and reduced the frequency of stool, IBS-C patients also suffer
from bloating, straining and incomplete sense of evacuation [1,4]. Because traditional therapies have poor efficacy on the main
symptoms of IBS-C patients, more treatment options will be valuable [6].
Linaclotide is a 14-amino acid peptide approved by the FDA for the treatment of IBS-C, which not only accelerates intestinal transit
but also improves abdominal pain [8,9]. This peptide belongs to heat-stable enterotoxin analog of E. coli, which activates GC-C on the
surface of intestinal epithelium, resulting in increased level of cyclic guanosine monophosphate (cGMP). Increased cGMP could
activate cystic fibrosis transmembrane conductance regulator (CFTR). This activation results in the secretion of bicarbonate and
chloride into the lumen, increasing intracavital fluid secretion and accelerating intestinal transit [10, 11]. Although linaclotide is
effective for the treatment of IBS-C, there are some side effects such as dose-dependent diarrhoea, abdominal discomfort and
flatulence in clinical trials [5,6]. Therefore, it is still necessary to improve the activity of linaclotide based on drug molecule design
———

[12,13]. However, the PDB files of linaclotide remains unknown. For this reason, the determination of the three-dimensional structure
of linaclotide is of importance.
Racemic protein crystallography has been proven to be a powerful technique for crystallizing proteins [14-18]. The structures of
many difficult-to-crystallize peptides/proteins of different sizes were resolved by the racemic protein crystallography combined with
the protein chemical synthesis [19-24]. In this work, we report the chemical synthesis of natural linaclotide (L-type) and its
enantiomeric D-peptide (D-type) by using Fmoc solid phase peptide synthesis (Fmoc-SPPS) method. The X-ray crystal structure of
linaclotide was determined in 1.59 Å by racemic protein crystallography. The crystal structure showed that linaclotide has a tight,
three-beta turns spatial structure immobilized by three pairs of disulfide bonds.
Fig. 1. (a) Sequence comparison among STh, STp and linaclotide. (b) Synthetic route. HPLC chromatogram and mass spectra of L-reduced
linaclotide (c) and D-reduced linaclotide (d).
Linaclotide is a heat-stable enterotoxin analog of E. coli consisting of 14 amino acids with three pairs of intramolecular disulfide
bonds (C1-C6, C2-C10 and C5-C13) (Fig. 1a) [25]. The use of racemic crystallography for the crystallization of linaclotide needs to
obtain L-type and D-type proteins. To this end, we used Wang resin as a solid scaffold and DIC (N,N'-diisopropylcarbodiimide) as
condensing reagent to synthesize L-type and D-type linaclotide directly through Fmoc-SPPS [26]. After the completion of the synthesis,
the crude linaclotide was cleaved from the resin and the side chain protecting group was removed by using a cleaving reagent
(TFA/H2O/phenol/TIPS = 87.5/5/5/2.5, v/v/v/v) (Fig. 1b) [27-42]. The L-type and D-type crude peptides were analyzed by analytical
HPLC and verified by ESI-MS. As show in Fig. 1c/1d, both crude peptides had good purity. After purification by RP-HPLC and
lyophilized, we obtained L-type and D-type reduced linaclotide (isolated yield: 44.9% for L-reduced linaclotide and 51.6% for D-
reduced linaclotide).

Fig. 2. a) The HPLC traces of folding of L-reduced linaclotide. b) The HPLC traces of folding of D-reduced linaclotide. c) HPLC
chromatogram and mass spectrum of purified L-linaclotide and D-linaclotide.
We then folded the L- and D-type reduced linaclotide by using a redox pair consisting of oxidized glutathione (GSSH) and reduced
glutathione (GSH). Both L- and D-type reduced linaclotide were first dissolved in 50 mmol/L tris buffer, respectively, then GSSH and
GSH were added to each buffer (molar ratio: peptide/GSSH/GSH = 1/10/100). The pH of the buffer was adjusted to 8.0 and folding
process was performed overnight at 25 °C [43]. As shown in Fig. 2a/2b, the L- and D-type reduced linaclotide were almost completely
converted to the corresponding L- and D-type linaclotide. After folding, the pH of the folded solution was adjusted to 2.0, then purified
by HPLC immediately to give L- and D-type linaclotide (isolated yield: 51.7% for L-linaclotide and 59.2% for D-linaclotide).
Fig. 3 a) CD spectra of L-linaclotide and D-linaclotide. b) SDS-PAGE of L-linaclotide and D-linaclotide.

Fig. 4. a) Crystal structure of racemic crystal of L-linaclotide and D-linaclotide. b) Views of L-linaclotide and D-linaclotide crystal structure. c)
Comparison of L-linaclotide and STP mimic (PDB: 1ETN). L-linaclotide, D-linaclotide and STP mimic are shown in orange, blue and cyan,
respectively.
With L- and D-type linaclotide in hands, we first verified the difference in secondary structure between L-type and D-type linaclotide
by circular dichroism (CD) spectra. As show in Fig. 3a, the CD spectrum of L-type linaclotides indicates a negative and a positive peak
in the range of 205-215 nm and 195-205 nm, respectively, while the curve of D-linaclotide is symmetrically opposite to the L-
linaclotide. SDS-PAGE analysis (Fig. 3b) shows that L-type and D-type linaclotide appeared between 17 and 26 kDa with a diffuse
appearance. These results imply that the structure of linaclotide was sufficiently rigid, which could not be completely denatured under
SDS conditions.
After confirming the protein characterization, we carried out the racemic crystallization experiment to obtain the X-ray structure of
linaclotide. Crystals were screened using a Hampton crystallization kit in 18 °C crystal chamber after mix the L- and D-type linaclotide
to their respective final concentrations of 10 mg/mL. After three days, we obtained the racemic crystals of linaclotide. The crystals
were grown in 0.2 mol/L potassium sodium tartrate tetrahydrate, 20% (w/v) polyethylene glycol 3,350 to form a P1 space group with
diffraction resolution of 1.59 Å. Structural information of linaclotide was obtained after molecular replacement and refinement.
As shown in Fig. 4a/4b, the crystals of L- and D-type linaclotide pack together and are almost mirror-image linked together.
Linaclotide stabilizes its spatial structure by three pairs of intramolecular disulfide bonds, giving it a compact spatial structure
containing three-beta turns. Linaclotide has a similar amino acid sequence to heat-stable enterotoxins, which is a class of diarrhea-
causing toxins secreted by intestinal pathogens. Through the structural comparison between linaclotide and heat-stable enterotoxin of E.
coli (STP mimic), we conclude that linaclotide has similar spatial arrangement (Fig. 4c) [25,44]. The acquisition of linaclotide crystals
potentially provides the basis for the subsequent optimization of the activity.
In summary, we obtained L- and D-type of linaclotide by protein chemical synthesis and acquired the X-ray crystal structure of
linaclotide for the first time through racemic crystallization technique. The structure of linaclotide forms a compact spatial structure
containing three-beta turns through three intramolecular disulfide bonds, which has a similar spatial arrangement to heat-stable
enterotoxin of E. coli (STP mimic). We anticipated that the X-ray crystal structure of linaclotide provides the basis for the optimization
and improvement of the activity of linaclotide. Our work also highlights the power of chemical protein synthesis in obtaining peptide
drugs for biochemical and structural studies.
Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 21572043) and the Fundamental Research
Funds for the Central Universities (No. PA2017GDQT0021).
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