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The Interplay of Disulfide Bonds, #-Helicity, and Hydrophobic Interactions Leads to Ultrahigh Proteolytic Stability of Peptides Yaqi Chen, Chaoqiong Yang, Tao Li, Miao Zhang, Yang Liu, Marc A. Gauthier, Yibing Zhao, and Chuanliu Wu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00567 • Publication Date (Web): 09 Jul 2015 Downloaded from http://pubs.acs.org on July 12, 2015
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The Interplay of Disulfide Bonds, α-Helicity, and Hydrophobic Interactions Leads to Ultrahigh Proteolytic Stability of Peptides
Yaqi Chen1†, Chaoqiong Yang1†, Tao Li1, Miao Zhang1, Yang Liu1, Marc A. Gauthier2, Yibing Zhao1, Chuanliu Wu1*
1
: State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, P.R. China 2
: Institut National de la Recherche Scientifique (INRS), EMT Research Center, 1650 boul. Lionel-Boulet, Varennes, J3X 1S2, Canada
†
These authors contributed equally
*To whom correspondence should be addressed, Email:
[email protected] 1 ACS Paragon Plus Environment
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Abstract The contribution of noncovalent interactions to the stability of naturally occurring peptides and proteins has been generally acknowledged, though how these can be rationally manipulated to improve the proteolytic stability of synthetic peptides remains to be explored. In this study, a platform to enhance the proteolytic stability of peptides was developed by controllably dimerizing them into α-helical dimers, connected by two disulfide bonds. This platform not only directs peptides towards an α-helical conformation, but permits control of the interfacial hydrophobic interactions between the peptides of the dimer. Using two model dimeric systems constructed from the N-terminal α-helix of RNase A and known inhibitors for the E3 ubiquitin ligase MDM2 (and its homologue MDMX), a deeper understanding into the interplay of disulfide bonds, α-helicity, and hydrophobic interactions on enhanced proteolytic stability was sought out. Results reveal that all three parameters play an important role on attaining ultrahigh proteolytic resistance, a concept that can be exploited for the development of future peptide therapeutics. The understanding gained through this study will enable this strategy to be tailored to new peptide, because the proposed strategy displays substantial tolerance to sequence permutation. It thus appears promising for conveniently creating prodrugs composed entirely of the therapeutic peptide itself (i.e., in the form of a dimer).
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Introduction As a class of molecule, peptides are promising therapeutic agents due to the availability of screening tools for identifying leads from ultra-high diversity libraries and their precedence for inhibiting protein–protein interactions.1-6 However, the inherent instability of peptides towards proteolytic digestion in the body is a major obstacle to their performance, mainly due to the functional diversity and aggressivity of endogenous proteases.7-10 Many strategies have thus been developed to improve resistance of peptides to proteolysis, including backbone/side-chain modification, unnatural residue substitution, and cyclization.10-14 These approaches, however, can be synthetically laborious or awkward to implement. As a consequence, simple strategies to effectively stabilize peptides from enzymatic digestion that do not have recourse to unnatural amino acids, extensive post-translational chemistry, or formulation strategies are of exceptional interest. In this context, Nature has itself evolved to protect naturally occurring peptides from enzymatic digestion in biological environments. For instance, cyclotides and defensins exploit N- to C- cyclization and/or regiospecific disulfide pairing to reduce susceptibility to enzymatic digestions.15-18 These strategies have been emulated in artificial peptide constructs.19-22 While protease resistance has been mainly thought to arise from reduced structural flexibility due to covalent cyclization, hydrophobic interactions and salt bridges are increasingly thought to be important.23-26 Indeed, they are well-known to play a central role in maintaining the structural integrity of proteins and imparting proteolytic stability. A particularly noteworthy example is the toxic amyloid fibrils, which are formed by the self-assembly of β-strand peptides via inter-strand hydrophobic interactions, and which are highly resistant to intracellular proteases.27 However, how noncovalent interactions between amino acid residues can be rationally manipulated to improve the proteolytic stability of synthetic peptides 3 ACS Paragon Plus Environment
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remains to be explored. In this study, we develop a platform to enhance the proteolytic stability of peptides by controllably dimerizing them into α-helical dimers, connected by two disulfide bonds. This platform not only directs peptides towards an α-helical conformation, but can also be exploited to modulate the interfacial hydrophobic interactions between the peptides of the α-helical dimer. Using two model dimeric systems constructed from the N-terminal α-helix of RNase A and known inhibitors for the E3 ubiquitin ligase MDM2 (and its homologue MDMX), a deeper understanding into the interplay of disulfide bonds, α-helicity, and hydrophobic interactions on enhanced proteolytic stability is sought out. Results reveal that these three parameters play important roles on attaining ultrahigh proteolytic resistance, a concept that can be exploited for the development of future peptide therapeutics.
Experimental Section Materials. The investigated peptides, N-terminally acetylated and C-terminally amidated, were supplied by Sangon Biotech (Shanghai, China) or ChinaPeptides (Shanghai, China) at >95% purity. All peptides were supplied with analytical chromatograms and mass spectra to confirm the identity and purity. MCF7 breast cancer cell line was purchased from CoBioer Biosciences Co., Ltd (Nanjing, China). Dulbecco’s modified eagle media (DMEM) was obtained from Thermo Scientific (Beijing, China). Other chemicals were purchased from major suppliers such as Sigma-Aldrich (Beijing, China), J&K Chemical (Guangzhou, China), and Sinopharm Chemical Reagent (Shanghai, China), and used as received. Millipore ultrapure water was used throughout the experiments. Instruments. Analytical and semi-preparative HPLC was performed by using a SHIMADZU system 4 ACS Paragon Plus Environment
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equipped with a prominence LC-20AD solvent delivery unit, a prominence DGU-20A3R degassing unit, a prominence SIL-20A autosampler, a prominence CTO-20A column oven and a prominence SPD-M20A photodiode array detector. A Bruker MicroFlex MALDI-TOF and a Bruker Esquire 3000plus ion trap ESI mass spectrometry were used for identifying isolated peptides. CD spectra were recorded with JASCO J-810 CD spectrometer. Synthesis of peptide dimers. 3=3, 6=6, 9=9 and 10=10 were prepared by oxidation in 100 mM phosphate buffer (pH 7.4) with 40 vol% TFE and purified by semi-preparative HPLC (1.0 mL·min–1 flow rate, isocratic with 5 vol% acetonitrile (ACN) + 0.1% trifluoroacetic acid (TFA) for 5 min followed by a linear gradient of ACN + 0.1% TFA (5–60 vol%) over 30 min; 67%, 35%, 64% and 84% yield, respectively). 7=7 was prepared by oxidation in aqueous NaOH solution (3 mM, pH ~9.5) with 50 vol% dimethyl sulfoxide (as both the oxidant and co-solvents for helix stabilization), following by purification with semi-preparative HPLC (80% yield). Though folding-and-stapling 7 through direct oxidation only produced the antiparallel dimer, parallel dimer of 7 can be obtained with a relatively low yield (~10%) if the cysteine residues of 7 was first activated by 2,2’-dithiopyridine, and then reacted with an another TFE folded 7. In addition, 8=8 and 11=11 were also prepared through this activation-based strategy. In a typical experiment, 0.1 mM peptide and 1.0 mM 2,2'-dithiodipyridine were first co-incubated in 100 mM phosphate buffer (pH 7.4) with 40 vol% TFE. Then, the activated peptide (both cysteines in peptides were activated by 2,2'-dithiodipyridine) was purified by HPLC. After that, the peptide 7, 8 or 11 (100 µM) and activated peptide (100 µM) were co-incubated in 100 mM phosphate buffer (pH 7.4) with 40 vol% TFE to form the dimer. Isolated peptide dimers were identified by MALDI-TOF mass spectrometry (Fig. S1, S7–S12), lyophilized and stored at –20 °C until used. 5 ACS Paragon Plus Environment
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Circular Dichroism (CD) Spectroscopy. CD spectra were recorded at room temperature (20 °C) using a 0.1 cm path length cuvette. The spectra were recorded in wavelength range of 190‒260 nm and averaged over 3 scans with a resolution of 1 nm, a bandwidth of 2 nm and a response time of 8 s. The sensitivity and the speed of the spectrometer were set to 100 mdeg and 50 nm·min–1, respectively. The baseline signal (phosphate buffer only) was subtracted from each spectrum. All peptides were dissolved in aqueous or mixed phosphate buffers to reach a concentration of 30 µM. Digestion analysis of disulfide pairing. A certain amounts of peptide dimers were dissolved in 0.25 mL phosphate buffer (100 mM, pH 7.4), which were then digested by the addition of 50 µL aqueous trypsin or chymotrypsin (0.1 mg·mL–1) for 30 min at room temperature. The digested fragments were isolated by HPLC and analyzed by MALDI-TOF or ESI mass spectrometry. Digestion kinetics analysis. All digestion reactions were performed in phosphate buffer (100 mM, pH=7.4) at room temperature (peptide concentration: 50 µM). Note that, for the enzymatic digestion of 6–8 and 11, and their dimers, ~4% DMSO was contained in phosphate buffers, because of the low solubility of these peptides in water and the stock solutions was first prepared in DMSO solvent. We have confirmed that 4% DMSO in buffers does not affect the kinetics of peptide digestion. Aliquots were taken from the samples at predefined times, and were immediately treated with 10% HPO3 to quench the reactions. The samples were then monitored chromatographically using a HPLC (water/acetonitrile with 0.1% TFA) and the digestion kinetics were calculated by integration of the 280 nm signal. Cell culture and cell-viability assay. MCF7 cells were maintained in DMEM media supplemented with 10% FBS and 1% penicillin/streptomycin. A total of 10000 cells/well were plated into 96-well cell culture dishes and incubated at 37 ℃ in a humidified atmosphere (95% RH) containing 5% CO2. 6 ACS Paragon Plus Environment
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After
24
h,
the
media
was
removed
and
DMEM
media
supplemented
with
1%
penicillin/streptomycin and different concentrations of peptides (0–10 µM) was added. After 20 h incubation, the media was removed and fed with 90 µL fresh media and 10 µL CCK-8 per well. After incubation for 2 h, the absorbance at 450 nm was measured using a plate reader. All assays were performed in triplicate, and percent cell viability was calculated by normalizing the data to vehicle treated controls. Reduction of antiparallel 7=7 in redox buffers. The solvents used to prepared the reducing stock solutions were first treated with N2 for 0.5 h. The thiol-disulfide exchange (i.e., reduction) reactions were performed in phosphate buffer (100 mM, pH=7.4) at room temperature in anaerobic incubator. Antiparallel 7=7 (50 µM) was incubated with 10 mM GSH, 10 mM DTT, mixture of 10 mM GSH and CGGC-containing peptide, and mixture of 10 mM GSH and CGC-containing peptide, respectively. Aliquots were taken from the samples at predefined times and were immediately treated with 10% HPO3 to quench the reactions. The samples were analyzed chromatographically by using HPLC, and the percentages of 7 and antiparallel 7=7 were calculated from the HPLC peak areas.
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Results and Discussion Development of the fold-and-staple strategy There are several instances in Nature in which disulfide bonds within proteins stabilize the α-helical conformation of some of their domains.18,28,29 However, isolated short peptides (ca.
