Amylin peptide hormone activates amylin receptors in brains and controls blood glucose and appetite. An amylin receptor activator pramlintide was developed for diabetes treatment. Currently, the amylin receptor activator with once-weekly injection has been tested for body weight reduction to treat obesity. Human amylin peptide was reported to form aggregates, while rat amylin has been shown soluble in aqueous solution. Here, multiple peptide activators for human amylin receptors were developed by introducing comprehensive mutagenesis to rat amylin peptide. The rat amylin peptide C-terminal fragment is known for interacting with human amylin receptor extracellular domain. The rat amylin peptide C-terminal fragment with eleven amino acids was used to screen for affinity-enhancing mutations. Up to twelve mutational combinations were found to significantly increase peptide affinity for amylin receptor extracellular domains by over 100-fold. Using these affinity-enhancing mutations, three representative rat amylin analogs with thirty-seven amino acids were made to test the potency increase for amylin receptor activation. All three mutated rat amylin analogs showed significant potency increases by 5- to 10-fold compared to endogenous rat amylin. These mutated peptide activators also showed higher potency for human amylin receptor activation than a clinical drug pramlintide. These amylin receptor activators developed in this study can be useful for the drug development targeting diabetes/obesity treatment.
Obesity is a common and serious disease worldwide with limited pharmacological options. Obesity is a leading contributor to type II diabetes development and it also induces multiple complications such as cardiovascular diseases and joint pain. Peptide hormone analogs that activate glucagon-like peptide 1 (GLP-1) receptors were recently approved for obesity treatment. GLP-1 receptor activators originally have been approved for type II diabetes by increasing insulin secretion. Among them, liraglutide and semaglutide were reported to reduce body weight and most recently once-weekly injection of semaglutide was approved for obesity treatment.
In addition to GLP-1, two more peptide hormones have shown potential for obesity treatment: glucose-dependent insulinotropic polypeptide (GIP) and amylin. GIP stimulates insulin secretion and is believed to reduce appetite. Once-weekly injection of a GIP and GLP-1 receptor dual agonist tirzepatide showed promising results for obesity treatment and its use for body weight control was recently approved in US.
Amylin is secreted from pancreas with insulin and amylin receptor activation controls blood glucose and body weight. An amylin analog pramlintide has been approved for diabetes treatment. Recent clinical trials with once-weekly injection of an amylin receptor activator cagrilintide alone and cagrilintide combination with a GLP-1 receptor agonist semaglutide showed a significant 10 to 17% body weight reduction.
The amylin receptor is the heterodimer complex of the calcitonin receptor and an accessary protein called receptor activity-modifying protein (RAMP). There are three types of RAMP in humans (RAMP1–3), and the calcitonin complex with each of RAMPs constitutes amylin receptor 1–3. Recent cryo-electron microscopy (cryo-EM) structures of the amylin receptors clearly showed the binding mode of peptide hormone amylin and calcitonin for their receptors. The N-terminal fragment of amylin interacts with the transmembrane domain of the calcitonin receptor. In contrast, the C-terminal fragment of amylin interacts with the extracellular domains of the calcitonin receptor and RAMP.
Human amylin peptide was reported to form aggregates that were visualized as amylin fibrils by cryo-EM. However, rodent amylin has a different amino acid composition compared to human amylin and it has been shown soluble in aqueous solution. Especially, rat amylin has been commonly used for animal studies and for cell signaling assay to activate amylin receptors. This study focuses on the design of potent rat amylin analogs that may enhance anti-obesity efficacy through amylin receptor activation. The hypothesis for this study was that introducing mutations to the peptide fragments can produce novel peptide ligands with altered (preferentially enhanced) affinity and potency for the amylin receptors. The primary endpoints of this study were to find affinity-enhancing mutations by at least 10-fold and to develop potency-enhancing peptide activators by 3-fold when compared to endogenous rat amylin. This study measured the binding affinity of the rat amylin peptide C-terminal fragment for purified human amylin receptor extracellular domains with fluorescence polarization peptide-binding assay. Comprehensive mutagenesis was introduced to the rat amylin peptide C-terminal fragment to find affinity-enhancing mutations. The resulting affinity-enhancing mutations were used for potency enhancement for human amylin and calcitonin receptor activation.
Dulbecco’s Modified Eagle Medium (DMEM) including 4.5g/L glucose, L-glutamine, and sodium pyruvate was purchased from Corning (Mediatech, Inc., Manassas, VA) to culture mammalian cells. The mixture of non-essential amino acids (NEAA, 100X) was purchased from Lonza (Basel, Switzerland). For mammalian cell culture, fetal bovine serum (Cat.# F2442) was purchased from Sigma-Aldrich (St. Louis, MO). NEBuilder® HiFi DNA Assembly Master Mix and several restriction enzymes were purchased from New England Biolabs (Ipswich, MA) and were used for cloning plasmid DNA constructs. 4–12% Mini-PROTEAN TGX Stain-Free Precast Gels were purchased from Bio-rad (Hercules, CA). All other reagents were purchased from Sigma-Aldrich, unless otherwise noted.
Human embryonic kidney (HEK) 293T cells and HEK293S GnTI- cells were purchased from ATCC (Manassas, VA) to express calcitonin and amylin receptor extracellular domains. HEK293T cells were also used to assess calcitonin and amylin receptor activation by using bioluminescence resonance energy transfer (BRET) assay. This BRET assay was well-established with HEK293 cells in a previous study and using one cell-line for BRET assay was assumed reasonable. The cell lines used for this study have been authenticated from the provider ATCC and the cells with the passage number less than 30 were used for this study. One Shot™ OmniMAX™ 2 T1R Chemically Competent E. coli cells (Cat.# C854003) were purchased from Invitrogen for DNA cloning.
Previously reported pHLsec-based vectors were used to express calcitonin and amylin receptor extracellular domains as secreted proteins. The following plasmid DNA constructs used in this study were previously described: pHLsec/hCTR.34–141-H 6 (H-pSL003), pHLsec/hRAMP1.24–111-(GSA)3-hCTR.34–141-H 6 (H-pSL005), pHLsec/hRAMP2.55–140-(GSA)3-hCTR.34–141-H 6 (H-pSL006), and pHLsec/hRAMP3.25–112-(GSA)3-hCTR.34–141-H 6 (H-pSL001). The following constructs for bioluminescence resonance energy transfer (BRET) assay that assesses G protein recruitment to G protein-coupled receptors were generously gifted from Dr. Nevin Lambert (Medical College of Georgia): NES-Venus-mGs (H-pSL028) and Nluc-N1 (H-pSL033). The cDNA sequence of full-length human calcitonin receptor was purchased from CDNA.org (Cat.# CALCR00000, H-pSL053). The cDNA sequences of human receptor activity-modifying protein 1 (RAMP1) (NM_005855.4, H-pSL056), RAMP2 (NM_005854.3, H-pSL042) and RAMP3 (NM_005856.3, H-pSL043) were purchased from Genscript (Piscataway, NJ, USA). The DNA construct of the calcitonin receptor C-terminally fused with NanoLuc luciferase (Nluc_FL_CTR (H-pSL054)) was generated for the current study using DNA Assembly reaction. Coding sequences of the above expression vectors were confirmed with Sanger sequencing performed by Psomagen (Rockville, MD). DNA expression plasmids were extracted and were purified from bacterial cells with NucleoBond® Extra Midi Plus kit (Macherey-Nagel, Germany) and they were stored at -20°C until their use for transient transfection into mammalian cells.
The general expression procedure of the calcitonin receptor (CTR) extracellular domain (ECD) alone and RAMP1, -2, or -3 ECD fused with CTR ECD as functional amylin receptor ECDs was previously reported. Briefly, the amylin receptor ECDs were expressed from HEK293T or HEK293S GnTI- cells with transient transfection. The expressed receptor proteins were secreted into cell culture media for 4 days at 37°C. These media were collected and the secreted receptor proteins were purified. After initial dialysis, multi histidine-tagged receptor proteins were purified with immobilized metal affinity column chromatography and the peak fractions were followed by size exclusion column chromatography as previously described. The purified receptor proteins were dialyzed to storage buffer and they were stored at -80°C until their use for fluorescence polarization peptide binding assay. All purification procedures were performed at 4°C.
All peptide fragments used for fluorescence polarization (FP) peptide binding assay were custom-synthesized from Genscript (Piscataway, NJ, USA). Fluorescein isothiocyanate (FITC)-labeled salmon calcitonin (sCT) fragment(22–32) and FITC-labeled AC413(6–25) with Y25P mutation were custom-synthesized from Genscript (Piscataway, NJ, USA). These peptides were used as probes for FP peptide binding assay. Full-length rat amylin (Cat.#H-9475.0500, with HPLC purity above 96%) was purchased from Bachem (Torrance, CA, USA). Pramlintide (Cat.# SML2523-5MG) was purchased from Millipore Sigma (Burlington, MA, USA). Other rat amylin analogs were purchased from Biomatik (Wilmington, Delaware, USA). All peptides used in this study except rat amylin purchased from Bachem and Pramlintide purchased from Millipore Sigma were HPLC-purified with at least 85% purity by Genscript (Piscataway, NJ) or Biomatik (Wilmington, Delaware, USA). Mass spectrometry performed at Genscript or Biomatik confirmed the correct molecular mass of all synthesized peptides. The extinction coefficient of FITC (63,000 M−1 ·cm−1 at 495 nm, pH 7.0) was used to calculate the concentration of the FITC-labeled peptides. For other peptides, extinction coefficients of tryptophan, tyrosine, and/or cystine residues were used to determine peptide concentrations. The sequences of the peptides used in this study are shown in the Table 1.
The general procedure of FP peptide binding assay with saturation and competition binding formats was previously described. 10 nM FITC-labeled sCT(22–32) was used as the peptide probe for CTR ECD binding, while 10 nM FITC-labeled AC413(6-25) with Y25P mutation was used as the peptide probe for all RAMP ECD-CTR ECD fusion proteins. K D values of the FITC-labeled peptide probes used for receptor protein binding were obtained from a saturation binding format. K D of FITC labeled sCT(22–32) for CTR ECD was 500 nM and K D values of FITC-labeled AC413(6–25) Y25P for RAMP1 ECD-CTR ECD, RAMP2 ECD-CTR ECD, and RAMP3 ECD-CTR ECD were 77 nM, 67 nM, and 252 nM. FP binding assay with a competition binding format was established with a fixed concentration (10 nM) of the FITC-labeled peptide probe. The concentration of the purified receptor ECD protein equal to the average K D value was used for the competition binding assay. 500 nM CTR ECD, 77 nM RAMP1 ECD-CTR ECD fusion protein, 67 nM RAMP2 ECD-CTR ECD fusion protein, and 252 nM RAMP3 ECD-CTR ECD fusion protein were used as K D values for competition binding assay. Receptor concentrations equal to the K D value produced approximately 50% of maximum anisotropy. Varying concentrations of a competitive peptide was used for the competition binding assay and the K I value of a competitive peptide was obtained from the competition binding curves. Non-linear regression equations used fo
