Synthesis of Stable Substance P Analog using Sunflower Trypsin Inhibitor Scaffold

Transcription

1 Synthesis of Stable Substance P Analog using Sunflower Trypsin Inhibitor Scaffold Abstract Substance P is a neuropeptide associated with pain and inflammatory signaling pathways. It is the ligand of neurokinin-1 receptor (NK1R). Some analogs of substance P that act as NK1R antagonist can be used as a drug to manage pain and inflammation. However, being a linear peptide they are not resistant to enzyme degradation and thus not orally active. To overcome this challenge, we synthesize a stable NK1R antagonist by grafting a linear 7-residue NK1R antagonist derived from the N-terminal sequence of substance P into the cyclic sunflower trypsin inhibitor-1 (SFTI-1) scaffold. This chimeric peptide SFSP(N) possesses two functions, trypsin inhibition and neurokinin-1 receptor (NK1R) inhibition. Trypsin inhibition is derived from trypsin inhibitory binding loop domain of cyclic sunflower trypsin inhibitor-1, while NK1R antagonism is derived from the N-terminal fragment of linear substance P. Consequently, the cyclic SFSP(N) is bifunctional and gains resistance against heating, ph and trypsin treatment. Based on the design and synthesis of stable NK1R antagonist adopting cyclized backbone and intramolecular disulfide constrains, we can prepare orally active peptidyl drugs to treat pain and inflammation. bacteria and plants [2], they can provide stability useful in drug engineering because of two structural features, cyclic backbone and intra-disulfide bridges. Both of these structural features are present in the synthesized SFSP(N) which contributes to its chemical and thermal stability. Figure 1 below is a showing the design of engineering SFSP(N) using the sequence of substance P N-terminal fragment and SFTI-1 trypsin inhibitor binding loop. The synthesized SFSP(N) consists of 16 amino acids in the sequence of CRPKPQQFCTKSIPPI. It possesses two functional loops which are trypsin inhibitory and NK1R inhibitory respectively. Trypsin inhibitory activity is derived from trypsin inhibitor binding loop of SFTI-1 and the NK1R inhibitory activity is derived from the substance P N-terminal fragment of linear substance P. Since SFTI-1 is a stable peptide for drug design due to its cyclic backbone and single intra-disulfide bridge [3], we envisioned that this grafted bifunctional cyclic peptide SFSP(N) can have both chemical and thermal stability and hence is orally active. Keywords Cyclic peptide, sunflower trypsin inhibitor- 1, trypsin inhibitory binding loop, substance P, substance P N-terminal fragment, pain relief, analgesic, SFSP(N) 1 INTRODUCTION Pain is unpleasant sensation that is triggered in the nervous system. It is an important stimulus to alert us of any physiological damage or disease, however undesirable chronic pain pose a significant concern to patients especially those with medical conditions such as trauma, diseases and post-surgery. Substance P is a linear peptide ligand that binds to neurokinin-1 receptor (NK1R) to act as a neuropeptide associated with pain and inflammatory signaling pathways. Certain analogs of substance P can act as NK1R antagonist which can be developed into a drug for the management of pain and anti-inflammatory effects [1]. The linear peptide form of substance P analog is metabolic unstable. To overcome this problem, we synthesize a stable NK1R antagonist based on cyclic sunflower trypsin inhibitor-1 (SFTI-1) and substance P to generate a metabolic stable cyclic peptide named SFSP(N). Cyclic peptides are found in mammals, Figure 1. Scheme of engineering of SFSP(N) from substance P N-terminal fragment and SFTI-1 trypsin inhibitor binding loop. 2 AIMS / OBJECTIVES Our objective is to synthesize a metabolic stable cyclic peptide, SFSP(N), by grafting substance P N-terminal fragment into the SFTI-1 secondary loop. This cyclic peptide should provide a stable scaffold for NK1R antagonist and trypsin inhibitory effect for potential treatment of pain and inflammation.

2 3 LITERATURE REVIEW / BACKGROUND Substance P is a neuropeptide associated with pain and inflammatory signaling pathways, functioning as a neurotransmitter and a neuromodulator [4, 5]. It is a small linear peptide composed of 11 amino acid residues derived from preprotachykinin A gene in chromosome 7 [6]. Substance P is important for the perception of pain and the transmission of pain information in the central nervous system (CNS). Upon pain stimulation, substance P from axon terminal of specific sensory neurons in the CNS is released into the synaptic cleft to bind to specific neurokinin-1 receptors (NK1R) for pain and inflammatory processes [1]. The linear N-terminal fragment of substance P can act as neurokinin-1 receptor antagonist reducing pain and inflammatory responses [7, 8]. However, it is easily degraded by digestive enzymes when taken orally due to its linearized form. Sunflower trypsin inhibitor-1, SFTI-1, is a natural cyclic peptide derived from sunflower seeds. It is the smallest and most potent Bowman-Birk trypsin inhibitor composing of 14 amino acids [9]. It consists of a trypsin inhibitor binding loop and a secondary loop. The cyclic nature of SFTI-1 makes it resistant to exopeptidase proteolysis and heat degradation. The loops are further structurally stabilized by a disulfide bridge between two cysteine residues within the peptide sequence [10, 11]. The trypsin inhibitor binding loop serves as a trypsin inhibitor to inhibit pancreatic trypsin degradation [12]. This allows it to withstand enzymatic cleavage in the digestive system. The secondary loop of SFTI-1 is not crucial for the trypsin inhibitory activity. Therefore it can be replaced by other bioactive peptides such as a pain-relief neuroreceptor antagonist. 4 METHODOLOGY AND/OR THEORETICAL APPROACH SFSP(N) was synthesized using SPPS and cyclized using the thia zip reaction. The sample was then purified and analyzed through various stability assays and trypsin inhibition assay. 4.1 SYNTHESIS OF PEPTIDE The synthesis of linear SFSP(N) peptide was done through solid phase peptide synthesis (SPPS). SPPS is a method for synthetic peptide synthesis pioneered by Robert Bruce Merrifield [13]. The overall synthesis scheme of SFSP(N) is summarized in Figure 2. In SPPS of SFSP(N), the C-terminal amino acid is covalently attached to the insoluble small solid resins via hydrazine linkers. Stepwise additions of fluorenylmethyloxycarbonyl chloride (Fmoc) protected amino acids are coupled to the growing peptide chain from the C-terminus to N-terminus. Figure 2. Synthesis scheme showing the process of synthesizing cyclic peptide, SFSP(N) An automated CEM Liberty synthesizer was used to synthesize SFSP(N). 2-chlorotritylchloride resin with substitution of 1.2 mmol/g was used as the solid support. One gram of resin was incubated overnight with µl hydrazine (NH 2 NH 2 ), 209 µl diisopropylethylamine (DIEA) and 5 ml dimethylformamide (DMF). It is then drained and rinsed twice for 10 minutes with methanol to block the remaining Cl -, it was then washed with DMF and DCM. This forms the hydrazine-trt(2-cl) resins which are resins bonded with hydrazine linkers for the first amino acid to bind. Isoleucine was the first C-terminal amino acid to be added to the resin hydrazine mg of Fmoc-Ile with mg 1-Hydroxy-7-azabenzotriazole in µl of DIC and 5 ml DMF was added to the prepared

3 resins. After mixing for 45 minutes, the resin is now coupled with Fmoc-Ile linked via hydrazine. It was then drain and vacuum dried for automated SPPS. Fmoc-Lamino acids in DMF were prepared for automated SPPS using CEM Liberty synthesizer with 25 ml activator (PyBOP), 307 ml deprotector (20% piperidine in DMF), 13 ml activator base consisting of 55% DMF, 35% DIEA and 10% dichloromethane (DCM). 4.2 CYCLIZATION OF PEPTIDE After synthesis of the linear peptide was completed, the covalently attached linear peptide was then cleaved from the resin using cleavage reagent containing 90% trifluoroacetic acid (TFA), 5% triisopropylsilane and 5% Milli-Q water for 2 hours. The precipitated with cold diethyl ether. This formed the linear SFSP(N) peptide hydrazide. This was followed by the cyclization of linear peptide using a 2 step method involving thia zip cyclization of the peptide hydrazide [14]. The first step was the conversion of the peptide hydrazide into peptide azide in aqueous sulphuric acid at ph 2 with 10 equivalents of 1 M NaNO 2 at 0 o C for 30 minutes. After 30 minutes, ph was adjusted to 7.5 using 1 M aqueous sodium hydroxide with 100 equivalents of methyl mercaptoacetate (MMA) at room temperature for 2 hours. This then initiated the second step involving the conversion of the peptide azide to thioester The thioester will undergo thia zip reaction to form the thiolactone, which then undergo acyl transfer to generate the cyclic SFSP(N) [14]. Oxidative folding of SFSP(N) to form disulfide bridge between cysteine side groups was done using 10% dimethyl sulfoxide (DMSO) overnight at room temperature to form the oxidative folded cyclic SFSP(N). Small scale analysis of the peptide products formed was carried out after each step by quenching with 0.1% TFA for reverse phase High-Performance Liquid Chromatography (HPLC) using a 2 x 100 mm C18 Vydac column. A linear gradient from 10% - 60% of 99.9% ACN with 0.1% TFA over 25 minutes was used. Aliquots from the peaks at 220 nm wavelength were collected and analyzed using Shimadzu mass spectrometer and Matrix-assisted laser-desorption ionization-time of flight mass spectrometry (MALDI- TOF MS). After identifying the correct retention time for SFSP(N) peak, large scale purification of SFSP(N) was then conducted using semi-preparative reverse phase HPLC with a 10 x 250 mm C18 Vydac column. A linear gradient from 10% - 60% of 99.9% ACN with 0.1% TFA over 80 minutes was used. The fractions of the SFSP(N) peaks were collected and analyzed using MALDI-TOF MS prior to lyophilization. 4.3 STABILITY AND INHIBITION ASSAYS The purified SFSP(N) was then subjected to heat, ph, trypsin stability assay and trypsin inhibition assays to determine its chemical and thermal stability Thermal Stability Assay SFSP(N) was subjected to a high temperature of 100 ⁰C over a period of 6 hours while the control was kept at room temperature. Samples were collected at regular time intervals and analyzed using reverse phase HPLC with a 2 x 100 mm C18 Vydac column. A linear gradient of 10% - 60% of 99.9% ACN with 0.1% TFA over 25 minutes was used. The retention time of the peaks was compared before and after a certain amount of time under heat treatment. The result for thermal stability assay is shown in Figure ph Stability Assay Low ph was used in this assay so as to mimic the acidic conditions of the human stomach since SFSP(N) is to be used as an orally administered drug. SFSP(N) was subjected to acidic conditions using ph 2 water adjusted by hydrochloric acid. The samples were collected and analyzed using reverse phase HPLC over a period of 6 hours. The methodology for reverse phase HPLC used is the same as in thermal stability assay and the results are shown in Figure Trypsin Stability Assay 100 µl solution of 25 µm SFSP(N) in ammonium bicarbonate (NH 4 HCO 3 ) buffer and 1 µl of 0.5 µg/µl trypsin was mixed into the solution and left at room temperature. 15 µl of samples were collected and analyzed over a 6 hours period using the same reverse phase HPLC methodology as thermal stability assay. The result of the trypsin stability assay is shown in Figure Trypsin Inhibition Assay 1mM of SFSP(N) was diluted to make various SFSP(N) concentrations (10 µm 1 nm) using 100 mm NH 4 HCO 3 as a buffer. 94 µl of the various SFSP(N) concentrations was pipetted into each well on a 96- wells plate followed by 1 µl of 0.5 µg/µl trypsin and 5 µl of 20 mm of N-α-benzoyl-DL-arginine-p-nitroanilide (BAPNA) to make up the total volume in each well to be 100 µl. SFSP(N) was not added to the blank and negative control. The positive control was done using SFTI-1 instead of SFSP(N). The plate was then incubated at 37 ⁰C for 30 minutes. A microplate reader was used to analyze its absorbance with measurement parameters set at 405 nm as the measured wavelength and 600 nm as the reference wavelength. The results of the absorbance was then analyzed to determine trypsin activity for the respective SFSP(N) concentrations used in the assay. A graph showing trypsin activity (%) against SFSP(N) log10

4 concentration in nm was plotted and the trend line was drawn to generate their respective linear equation to determine their half maximal inhibitory concentration (IC 50 ) of SFSP(N). The graph is shown in Figure 8. 5 RESULTS The synthesis of oxidative folded cyclic SFSP(N) was achieved and were found to be stable to heat, ph and trypsin over a period of 6 hours, the IC 50 of its trypsin inhibition was determined to be 0.56 µm. 5.1 Cyclization of Peptide Each steps of the cyclization process was monitored by reverse phase HPLC by analyzing samples of peptide hydrazide, reduced cyclic peptide and oxidatively folded SFSP(N) generated. It revealed that there was an expected shift in the retention time of the peaks observed at 220 nm wavelength. This was a result of a change in hydrophobicity of the respective peptides resulting in a change in the retention time observed in their HPLC profiles shown from the peaks in Figure 3 below. Figure 4. MALDI-TOF MS result of oxidative folded cyclic SFSP(N) (peak F) with a mass of 1823 Da 5.2 Thermal Stability Assay The thermal stability assay result of SFSP(N) in Figure 5 below showed that the aliquots collected over a regular time during the 100⁰C heat treatment had HPLC profiles with the same retention time and similar height and peak area over a period of 6 hours. No new peaks indicating SFSP(N) degradation was present. This suggests that SFSP(N) is stable to heat treatment. Figure 3. Analytical HPLC profile (220nm wavelength) of peptide hydrazide (peak H) with retention time of 13.5 min; reduced cyclic peptide (peak R) with retention time of 16.8 min; oxidative folded cyclic SFSP(N) (peak F) with retention time of 15.2 min; peak I indicates impurities Upon further analysis of the fraction collected after overnight oxidative folding using 10% DMSO, MALDI-TOF MS confirmed that the final synthetic product was indeed SFSP(N) as the molecular mass of the fraction collected was found to be 1823 Da which is consistent with the expected theoretical mass of SFSP(N). The MALDI-TOF MS result is shown in Figure 4. Figure 5. Thermal stability assay of SFSP(N) (peak F) in 100⁰C heat treatment with retention time of 15.3 min 5.3 ph Stability Assay ph stability result of SFSP(N) in Figure 6 also showed that the aliquots collected over a regular time from the ph 2 treatment had similar HPLC profiles over the 6 hours period with the same retention time and similar height and peak area. Therefore, this implied that SFSP(N) did not degrade in acidic conditions over time, indicating that it is stable to low ph conditions. This makes SFSP(N) ideal for potential oral drug treatment as it can withstand the acidic condition found in the stomach without having to lose its structure.

5 SFTI-1 trypsin inhibitor binding loop was responsible for the trypsin inhibition activity of SFSP(N) [7]. Figure 6. ph stability assay of SFSP(N) (peak F) in ph 2.0 with retention time of 15.3 min 5.4 Trypsin Stability Assay In Figure 7 below, the trypsin stability assay result of SFSP(N) similar showed similar HPLC profiles for the aliquots collected over 6 hours in the presence of trypsin. Their HPLC profiles had the same retention time and similar height and peak area. This proved that the cyclic nature of SFSP(N) can effectively withstand trypsin degradation in the digestive system and allowed SFSP(N) to be metabolic stable. Figure 7. Trypsin stability assay of SFSP(N) (peak F) with retention time of 16.2 min 5.5 Trypsin Inhibition Assay SFSP(N) contains the trypsin inhibitor binding loop that confers trypsin inhibition. Figure 8 shows the result of the trypsin inhibition assay. Based on the equation generated from the graph, the IC 50 of the trypsin inhibitory activity of SFSP(N) was at 0.56 µm. Past research have reported that the Lys5-Ser6 site of the Figure 8. Trypsin inhibition assay of SFSP(N) having a calculated IC 50 of 0.56 µm 6 DISCUSSION The peptide sequence of SFSP(N) was elongated on a hydrazine-resin by solid phase peptide synthesis using Fmoc chemistry. The C-terminal hydrazide group can be converted into a thioester. It was further reacted with the N-terminal Cys through the Cys-thioester ligation reaction to afford a macrocyclic backbone. The cyclization process was accelerated via the thia zip mechanism. In the presence of intramolecular Cys residues, their side chain thiols could interact with the C-terminal thioester to form thiolactone intermediates via transthioesterification. When N- and C-termini were linked together by a thioester bond, the S-N acyl shift occurred through a five-member transition to afford a new peptide bond, which resulted in an end-to-end cyclic peptide [15]. The purification of SFSP(N) using HPLC resulted in MMA dimer co-eluting with the oxidatively folded SFSP(N) as MMA dimer has similar hydrophobicity with the latter. This was overcome by reducing the MMA dimer with TCEP overnight to form MMA monomers that eluted earlier. Semi-preparative reverse phase HPLC was used to separate the cyclic SFSP(N) from the MMA monomers. Once purified, the cyclic SFSP(N) fraction collected was then oxidized using 10% DMSO to form oxidative folded SFSP(N). The various stability assays were conducted to prove that the cyclic nature of SFSP(N) peptide had allowed it survive harsh conditions under heat, low ph and trypsin treatments to retain its structure and hence its function. The results of the stability assays showed that the cyclic SFSP(N) was able to retain its structure and not degrade under high temperatures and low ph. SFSP(N) was also able to resist to trypsin cleavage. This allows SFSP(N) to possess both thermal and chemical stability for it to be metabolic stable.

6 The trypsin inhibition of SFSP(N) with IC 50 of 0.56 µm which may be less potent than that of SFTI-1 due to the grafted substance P N-terminal fragment in SFSP(N) that have replaced the secondary loop of the native SFTI-1. Studies have reported that the stable arrangements of cross-linking hydrogen bonds between the single disulfide bridge and β-strands of SFTI-1 contribute to the high trypsin inhibitory effect [9]. Therefore, the removal of the secondary loop from native SFTI-1 in SFSP(N) may have disrupted the stable arrangement of the hydrogen bonds, contributing to the decreased in trypsin inhibition effect. Hence the replacement of the secondary loop with substance P analog could have caused the weaker trypsin inhibition activity of the trypsin inhibitor binding loop in SFSP(N). Nevertheless, its ability to inhibit trypsin at relatively low concentrations makes SFSP(N) ideal for being used as a metabolically stable drug for pain management and anti-inflammation effects. 7 CONCLUSION We managed to synthesize SFSP(N) from substance P N-terminal fragment and SFTI-1 trypsin inhibitor binding loop. The synthesis of SFSP(N) was achieved from Fmoc SPPS and thia zip cyclization and oxidative folding by DMSO. Heat, low ph and trypsin degradation were tested on the synthesized SFSP(N), and it proved to be stable in these treatments. This makes SFSP(N) a metabolic stable bifunctional peptide. Therefore, based on the new SFTI-1 scaffold we developed, we can potentially develop orally active drugs to treat pain and inflammation. ACKNOWLEDGEMENT I would like to express my gratitude to my supervisor for allowing me to take on this project in his laboratory. Next, I want to give my sincere gratefulness to my mentors for being so patient, supportive and understanding in guiding and assisting me throughout the course of this project. I would like to thank the rest of the lab personnel for the pleasant and memorable experience working in the lab. REFERENCES 1. Gerard, N.P., et al., Human substance P receptor (NK-1): organization of the gene, chromosome localization, and functional expression of cdna clones. Biochemistry, (44): p Craik, D.J., Seamless Proteins Tie Up Their Loose Ends. Science, (5767): p Quimbar, P., et al., High-affinity Cyclic Peptide Matriptase Inhibitors. Journal of Biological Chemistry, (19): p Harrison, S. and P. Geppetti, Substance p. Int J Biochem Cell Biol, (6): p Datar, P., et al., Substance P: structure, function, and therapeutics. Curr Top Med Chem, (1): p Krause, J.E., et al., Three rat preprotachykinin mrnas encode the neuropeptides substance P and neurokinin A. Proc Natl Acad Sci U S A, (3): p Daly, N.L., et al., The Absolute Structural Requirement for a Proline in the P3 -position of Bowman-Birk Protease Inhibitors Is Surmounted in the Minimized SFTI-1 Scaffold. Journal of Biological Chemistry, (33): p Wiktelius, D., Z. Khalil, and F. Nyberg, Modulation of peripheral inflammation by the substance P N-terminal metabolite substance P1-7. Peptides, (6): p Korsinczky, M.L.J., et al., Solution structures by 1H NMR of the novel cyclic trypsin inhibitor SFTI-1 from sunflower seeds and an acyclic permutant. Journal of Molecular Biology, (3): p Colgrave, M.L., et al., Sunflower trypsin inhibitor-1, proteolytic studies on a trypsin inhibitor peptide and its analogs. Biopolymers, (5): p Legowska, A., et al., Implication of the disulfide bridge in trypsin inhibitor SFTI-1 in its interaction with serine proteinases. Bioorg Med Chem, (23): p Korsinczky, M.L., R.J. Clark, and D.J. Craik, Disulfide bond mutagenesis and the structure and function of the head-to-tail macrocyclic trypsin inhibitor SFTI-1. Biochemistry, (4): p Merrifield, R.B., Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. Journal of the American Chemical Society, (14): p Tam, J.P., Y.-A. Lu, and Q. Yu, Thia Zip Reaction for Synthesis of Large Cyclic Peptides: Mechanisms and Applications. Journal of the American Chemical Society, (18): p Hruby, V. and G. Gregg Bonner, Design of Novel Synthetic Peptides Including Cyclic Conformationally and Topgraphically Constrained Analogs, in Peptide Synthesis Protocols, M. Pennington and B. Dunn, Editors. 1995, Humana Press. p