To protect peptide pharmaceuticals against peptidases

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To protect peptide pharmaceuticals against peptidases ARTICLE in JOURNAL OF PHARMACOLOGICAL AND TOXICOLOGICAL METHODS · FEBRUARY 2010 Impact Factor: 2.39 · DOI: 10.1016/j.vascn.2010.02.010 · Source: PubMed

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Journal of Pharmacological and Toxicological Methods 61 (2010) 210–218

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Journal of Pharmacological and Toxicological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j p h a r m t ox

Original article

To protect peptide pharmaceuticals against peptidases R. Rink a, A. Arkema-Meter a,1, I. Baudoin a,2, E. Post a,3, A. Kuipers a, S.A. Nelemans b, M. Haas Jimoh Akanbi a,4, G.N. Moll a,⁎ a b

BiOMaDe Technology Foundation, Nijenborgh 4, 9747 AG Groningen, The Netherlands Molecular Neurobiology, University of Groningen, Biological Center, Kerklaan 30,9751 NN Haren, The Netherlands

a r t i c l e

i n f o

Article history: Received 1 December 2009 Accepted 16 February 2010 Keywords: Cyclization Lactococcus lactis Lantibiotic Methods NisB NisC Proteolytic Therapeutic peptide

a b s t r a c t Introduction: The major hurdle in the application and delivery of peptide pharmaceuticals is their rapid in vivo breakdown. Methods: We here combined two approaches to stabilize peptide pharmaceuticals, introduction of D-amino acids and cyclization, by applying an innovative enzymatic method. This method yields peptides with thioether bridges between a D-amino acid and an L-amino acid. On the basis of guidelines concerning the flanking residues of serines/threonines and cysteines, a peptide of interest is designed with serine/threonine and cysteine at appropriate positions to allow their effective participation in cyclization. In Lactococcus lactis the peptide of interest is directly or via a spacer genetically fused to a lantibiotic leader peptide which induces enzyme-catalysed synthesis of a thioether-bridged peptide. The peptide is translocated via a lantibiotic transporter, analysed by mass spectrometry and the leader peptide is removed. Because of its therapeutic relevance and terminal modifications we chose the decapeptide Luteïnizing Hormone Release Hormone (LHRH) as a test case for thioether bridge introduction. The N-terminal pyroglutamate protects against aminopeptidase activity; the amidated C-terminus, which occurs in 50% of all therapeutic peptides, precludes carboxypeptidase action and is essential for optimal receptor interaction. We had Lactococcus posttranslationally introduce a thioether bridge between residues 4 and 7 of the Leu7Cys-LHRH analog QHWSYGCRPG. The N-terminal glutamine of the thioether-bridged peptide could be converted in pyroglutamate. The introduction of the thioether bridge proved to be compatible with subsequent chemical and enzymatic amidation methods. In this way biologically produced thioether LHRH was compared with LHRH isomers obtained by base-assisted sulfur extrusion. Results: Biologically produced thioether LHRH is the most stable thioether LHRH isomer with strongly enhanced proteolytic resistance compared to natural LHRH. Discussion: The data convincingly demonstrate the broad perspective of stereo- and regiospecifically generating cyclized peptide pharmaceuticals with significantly enhanced therapeutic potential. © 2010 Elsevier Inc. All rights reserved.

1. Introduction Tremendous chemical and biological diversity occurs among peptide pharmaceuticals. Their high specificity has important clinical value. They are often very potent which makes them attractive for a ⁎ Corresponding author. Tel.: + 31 50 3638070; fax: + 31 50 3634429. E-mail addresses: [email protected] (R. Rink), [email protected] (A. Arkema-Meter), [email protected] (I. Baudoin), [email protected] (E. Post), [email protected] (A. Kuipers), [email protected] (S.A. Nelemans), [email protected] (M.H.J. Akanbi), [email protected] (G.N. Moll). 1 Present address: Department of Pathology and Laboratory Medicine, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. 2 Present address: University Medical Center Groningen, Department of Cardiology, Experimental Cardiology section, 9700 RB Groningen, The Netherlands. 3 Present address: Pharmacy, Antonius Deusinglaan 1, 9713 AV Groningen, Drug Targeting, The Netherlands. 4 Present addresses: Department of Pharmacology and Therapeutics, College of Health Sciences, University of Ilorin, Ilorin, Nigeria. 1056-8719/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.vascn.2010.02.010

therapeutic purpose. Peptide pharmaceuticals are also widely used in academic research. Peptide fragments of protein and polypeptides have been found with the functionality of an originally larger molecule. The simplicity of peptides makes them useful models to study domains of larger proteins and make them also suitable for drug design. Their small size allows them to penetrate into tissues that are not reached by larger proteins. These factors contribute to a significant growth in the application of peptide pharmaceuticals. The market of peptide pharmaceuticals is growing and they are nowadays being applied in all major disease fields including oncology, cardiovascular disease, diabetes, obesity, diagnostics, arthritis and central nervous system disorders (Ayoub and Scheidegger 2006). Peptide pharmaceuticals also have disadvantages. A major problem of peptide pharmaceuticals is their rapid degradation by proteolytic enzymes. Aminopeptidases and carboxypeptidases mediate breakdown starting at the peptide's termini, whereas a variety of endopeptidases can cleave distinct cleavage sites within the peptide. Peptide pharmaceuticals which have not been protected by formulation or

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modifications undergo proteolytic degradation in the gastrointestinal tract and liver which precludes oral delivery. Also after arrival into the circulation, peptides may undergo rapid breakdown. Proteolytic breakdown may be prevented or reduced by cyclization (Li and Roller 2002) and by the introduction of D-amino acids (Bessalle, Kapitkovsky, Gorea, Shalit & Fridkin, 1990; Hong, Oh & Lee, 1999). Here we present a biotechnological method which exploits Lactococcus lactis containing peptide-modifying enzymes. This approach leads to the stabilization of peptides by the simultaneous introduction of thioether bridges and D-amino acids. We applied this method to LHRH a decapeptide hormone with significant therapeutic relevance. LHRH has been subject of much research aiming at the development of potent agonists and antagonists for chemical inhibition of reproduction and antitumor agents (Mezö, Manea, Szabi, Vincze, & Kovacs, 2008). Natural LHRH has a pyroglutamate (pE) residue at position 1 and is amidated at the C-terminus. These modifications of the peptide's termini protect them against aminopeptidases and carboxypeptidases, respectively. In addition amidation of the carboxyterminus, which occurs in about 50% of all therapeutic peptides, is important for the receptor interactions of LHRH as for that of many other peptides. Neither pE formation nor amidation takes place within Lactococcus. Therefore, we investigated the feasibility of modifying the termini of the peptide after thioether bridge introduction and production by L. lactis. Both enzymatic and chemical methods were applied to amidate the C-terminus. We demonstrate that the presented stabilization method combined with modifications of the peptide's termini resulted in thioether-bridged LHRH analogs which are strongly resistant against proteolytic degradation. 2. Materials and methods 2.1. Materials Peptide QHWdCYGCRPG-NH2 (dC stands for D-cysteine) was obtained from Pepscan Lelystad, NL. Carboxypeptidase Y, antipain and leupeptin were purchased from Sigma (Zwijndrecht, NL). 2.2. Bacterial strains and plasmids

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amplification of the plasmid pNZnisA-E3 (Kuipers et al., 2004), using antisense primers with a complementary part to the nisin leader and a 5′ overhang, encoding the peptide sequences, and one universal phosphorylated sense primer annealing downstream the NisA peptide-coding sequence. Electrotransformation of L. lactis was carried out as described previously (Holo and Nes, 1995) using a Biorad gene pulser (Biorad, Richmond, CA). Nucleotide sequence analysis was performed by BaseClear (Leiden, NL). 2.4. Growth conditions L. lactis was grown at 30 °C in M17 broth (Terzaghi and Sandine 1975) supplemented with 0.5% glucose (GM17), chloramphenicol (5 μg/mL) and erythromycin (5 μg/mL) or in minimal medium (Jensen and Hammer 1993; Rink et al., 2005) supplemented with 1 μg/L nisin for induction. Cultures were grown on minimal medium after induction with nisin prior to sample preparation for mass spectrometry or peptide purification. 2.5. Method to stabilize peptides by enzymatic introduction of thioether bridges A thioether bridge was introduced into an LHRH analog by exploiting bacterial modification enzymes (Fig. 1) involved in the biosynthesis of nisin (Kluskens et al., 2005). The lantibiotic nisin is produced by some strains of Lactococcus lactis and contains five thioether bridges which are posttranslationally introduced. The nisin prepeptide consists of an N-terminal leader peptide of 23 amino acids, necessary for modification and export, followed by a 34 amino acid propeptide which can be posttranslationally modified. The dehydratase NisB dehydrates serines and threonines in the NisA propeptide and these dehydrated residues are subsequently regio- and stereospecific coupled to cysteines, which coupling is catalysed by the cyclase, NisC (Li et al., 2006; Rink, Kluskens, Kuipers & Driessen et al., 2007c). Nisin is exported out of L. lactis by the transporter NisT. We used the nisin modification and transport machinery to introduce a thioether ring in an LHRH analog and to secrete the stabilized analog into the medium. To this end we genetically made constructs encoding for the LHRH or hybrid nisin–LHRH sequences.

Strains and plasmids are listed in Table 1. 2.3. Molecular cloning PCR was performed with Phusion DNA polymerase (Finnzymes, Finland). Ligation was carried out with T4 DNA ligase (Roche). Restriction enzymes used for cloning strategies were purchased from Biolabs (New England). The different peptide constructs were made by

2.5.1. Design of a thioether-bridged LHRH analog Luteinizing hormone release hormone is a decapeptide with an Nand C-terminal modification. The N-terminus consists of pE and the C-terminus is amidated (Fig. 2AB). These modifications might provide some resistance against exopeptidases, yet they do not provide resistance against other peptidases/proteases for instance pyroglutamyl peptidase and endopeptidases. We here applied the nisin modification

Table 1 Bacterial strains and plasmids.

Strain NZ9000 ΔAcmA

Plasmids pIL253-derived pIL3BTC pNZ8048-derived pTP–LHRH1 pLPlhrh pTP–LHRH1A pTP–nisin–LHRH1 pTP–LHRH1CK pTP–LHRH1PCK pTP–LHRH1T

Characteristics/encoding

Reference

nisRK+

Kuipers, de Ruyter, Kleerebezem & de Vos, 1997; Buist et al., 1995

Cm-r MSTKDFNLDLVSVSKKDSGASPRQHWSYGCRPG MSTKDFNLDLVSVSKKDSGASPRITSISLCTPGCKTGALMGCNKQHWSYGCRPG MSTKDFNLDLVSVSKKDSGASPRITSISLCTPGCKTGALMGCNKQHWSYGCRPA Prenisin-QHWSYGCRPG MSTKDFNLDLVSVSKKDSGASPRITSISLCTPGCKTGALMGCNKQHWSYGCRPGCK MSTKDFNLDLVSVSKKDSGASPRITSIALCTPGCKTGALMGCNRQHWSYGCRPCK MSTKDFNLDLVSVSKKDSGASPRITSIALCTPGCKTGALMGCNRQHWTYGCRPG

Data in bold indicate sequences of LHRH analogs.

Simon and Chopin 1988 Rink et al., 2005 Kuipers, de Ruyter, Kleerebezem & de Vos, 1998 This study Kluskens et al., 2005 This study This study This study This study This study

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dimethylaminopyridinium tetrafluoroborate) modification was used to convert free cysteine residues to isothiocyanates in a 25 mM citrate buffer pH 3 (Rink et al., 2007c). Formation of the isothiocyanate part will result in a mass increase of 25 Da, whereas the absence of a mass shift indicates that the cysteine is involved in ring formation. The peptide samples (1 mg/mL) were allowed to react with 2 mg/mL CDAP in the presence of 10–20% acetonitril for 15 min at room temperature. The reaction with CDAP was preceded by 0.5 mg/mL triscarboxyethyl phosphine (TCEP) treatment to remove the cysteine additions which are often observed in peptides having cysteines that do not participate in intramolecular crosslinking. For Maldi-TOF, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, 1 μL of sample was spotted directly on target, dried, washed with 3 μL milliQ or 5% acetonitril, dried and overlayed with 1 μL 5 mg/ mL α-cyano-4 hydroxycinnamic acid in 50% acetonitril in 0.1% (v/v) trifluoroacitic acid. Mass spectra were recorded with a Voyager DE Pro Maldi-TOF mass spectrometer. In order to maintain high sensitivity, an external calibration was applied. 2.9. pE formation

Fig. 1. Lanthionine synthesis by the nisin modification enzymes. The dehydratase, NisB, dehydrates serine (or threonine) to yield dehydroalanine (or dehydrobutyrine). The cyclase, NisC, couples dehydroalanine (or dehydrobutyrine) to cysteine, yielding lanthionine (or methyllanthionine).

machinery for introducing a thioether bridge in an LHRH analog. For the posttranslational introduction of a thioether bridge a serine/threonine and a cysteine is required. Neither Ser4 nor Leu7 are considered to be essential for functionality (Sealfon, Weinstein & Millar, 1997). NisBcatalysed dehydration is favoured by the presence of directly flanking hydrophobic residues (Rink et al., 2005; Rink et al., 2007a). Ser4 is flanked by a Trp and a Tyr which will favour NisB-mediated dehydration of Ser4. Since no cysteine is present in LHRH Leu7 was replaced by Cys7 by mutagenesis. The flanking Gly6, which flanks Cys7, likely favours cyclization (Rink et al., 2005). 2.6. Trypsin treatment Those LHRH analogs, which were posttranslationally modified by the introduction of thioether bridges and produced by Lactococcus lactis, were cleaved from the nisin–LHRH hybrid by trypsin treatment. Typically, to 200 μL purified peptide (1 mg/mL), 25 μL 1 M Tris–HCl pH 8.0 and 10 μL 1 mg/mL trypsin were added followed by 15 min incubation at 37 °C and HPLC purification. 2.7. Non-enzymatic ring closure In vitro ring closure by base-assisted sulfur extrusion (Galande, Trent & Spatola, 2003) from 1 mg/mL disulfide bridged peptide, QHWdCYGCRPG-NH2, was performed by increasing the pH to 11 with ammonia and incubating samples overnight at 37 °C (Fig. 2C). Ammonia was removed by speed-vacuum drying of the modified peptide. The dried peptide was subjected to pE formation (see Section 2.9) and the desired modified peptides were purified to homogeneity by HPLC. 2.8. Mass spectrometry Peptides were purified from the medium fraction by ziptip purification (C18 ziptip, Millipore) or by subjecting medium volumes up to 1 mL to zipplate (Millipore) purification. The CDAP (1-cyano-4-

Complete conversion of glutamine to pyroglutamate (pE) was induced by incubation of the peptide during 6 h in 10 mM NaH2PO4 at pH 5 at 60 °C. The desired peptide was purified by HPLC. pE formation resulted in a mass decrease of 17 Da and a 2 min shift in HPLC retention time. 2.10. Enzymatic amidation A reaction mixture typically containing 126 μL 0.5 mM peptide, 125 μL 3 M glycinamide, 25 μL 1 M Tris–HCl pH 8.0, 2 μL 0.5 M EDTA and 5 μL of 1 mg/mL carboxypeptidase Y. Peptides were either pEHW [AYGA]cRPA or pEHW[AYGA]cRPG and were incubated at 37 °C. [A–A]c stands for lanthionine: (D)Ala-S-Ala. Samples of 5 μL were taken after 0, 10 and 50 min, quenched in 1 mL milliQ and analysed by HPLC. 2.11. Chemical amidation Amidation was performed following a previously described method (Nakagawa et al., 1994). pEHW[AYGA]cRPGCK was incubated during 15 min at room temperature with 10 μL 25 mM citrate buffer pH 3.0, 1 μL of 10 mg/mL Tris[2-carboxyethyl]phosphine (TCEP) after which 2 μL 10 mg/mL CDAP was added and the reaction mixture was incubated for 15 min at room temperature. Backbone cleavage liberating the amidated peptide was obtained by incubation during 10 min at room temperature with 3 M ammonia. The reaction mixture was dried by speed-vac and the desired end-product was isolated and purified by HPLC. Alternatively, methyl, ethyl and propylamine extensions were prepared by using the QHWSYGCRPCK-derived pEHW[AYGA]cRPCK variant (lacking G10). This was executed as described above but instead of using 3 M ammonia, 1 M of methyl-, ethyl- or propylamine final concentration was used. In this way the final peptide obtained methyl-, ethyl- or propylamine extensions replacing the C-terminal G. Peptides were purified by HPLC. 2.12. Purification of biologically produced peptides Medium supernatant from the desired production strain of L. lactis grown in MEM medium was passed through a 0.45 μm filter, and equilibrated with 1 volume 100 mM lactic acid, pH 2.5. Peptide was bound to a 5 mL HiPrep SP column, washed with 50 mM lactate buffer pH 4 and eluted with 50 mM lactate buffer containing 1 M NaCl. The eluate was desalted by passage over a PD-10 gel filtration column. The eluted peptide was subsequently freeze-dried. The LHRH peptide was digested by trypsin and purified by HPLC. The peptide was

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Fig. 2. ABC. Synthesis of thioether–LHRH. A) Structures of natural LHRH (upper) and D,L 4,7 thioether-bridged LHRH 11 (lower). B) Stereospecific enzymatic introduction of a thioether bridge in a precursor peptide followed by chemical modification of its termini. C) Introduction of a thioether bridge without stereo- and regiospecificity by base-assisted sulfur extrusion from a disulfide-bridged peptide.

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subsequently subjected to pE formation and amidation reactions as described in Sections 2.9–2.11.

area was compared to control and was taken as a measure of the peptide degradation.

2.13. HPLC purification of peptides

3. Results

Peptides were purified on a HP1050 HPLC system, using a Vydac C18 5µ 250 × 4.6 mm column. The peptides were eluted with a gradient of buffer A (0.1% trifluoroacetic acid (TFA) in milliQ) and buffer B (0.1% TFA in acetonitril). The gradient used was 10% to 50% buffer B with a slope of 1%/min. Peaks were collected and dried under vacuum using a speed-vac apparatus. The amount of peptide was determined by measuring the absorption at 280 nm from a peptide solution prepared in milliQ. The concentration was calculated using the molar absorption coefficient of 7.12 mM− 1 cm− 1.

3.1. Optimalization of production of a nisin–LHRH hybrid

2.14. 1H-NMR analysis of LHRH peptides The differences between LHRH1, LHRH11 and LHRH12 were assessed by homonuclear proton NMR. LHRH1 (0.8 mM), LHRH11 (3.1 mM) and LHRH12 (1.6 mM) were dissolved in DMSO-d6 and 2DTOCSY and 2D-NOESY spectra were recorded at 500 MHz at room temperature. 2.15. Preparation of protease-rich pig-tissue homogenates Pancreas mixture was obtained from Sigma. Pancreas contains mainly trypsin and has less of the other kinds of proteases than liver and kidney cortex. Kidney inner medulla membranes were isolated essentially as described previously (Booth & Kenny 1974). Kidney medulla was dissected and homogenized in a blender at full speed for 2 min in 10 volumes of 10 mM mannitol, 2 mM Tris–HCl, pH 7.1. The homogenate was centrifuged for 2 min at 200 × g, MgCl2·6H2O was added to 10 mM followed by stirring during 15 min on ice. The homogenate was centrifuged at 1500 × g at 4 °C for 12 min. The supernatant was centrifuged at 15,000 × g for 12 min. The pale-pink layer on top of the pellet was resuspended in 0.5 volume of 10 mM mannitol, 2 mM Tris–HCl pH 7.1 and MgCl2∙6H2O was added to 10 mM. The suspension was centrifuged at 2200 ×g for 12 min and the supernatant was centrifuged at 15,000 ×g for 12 min. The pellet, containing inner medulla membranes is resuspended in 0.05 volume. Isolation of liver membranes was performed essentially as previously (Dickey, Fishman, Fine & Navarro, 1987), all steps being at 4 °C. Fresh liver was weighed, placed in 4 volumes of ice-cold homogenization buffer, 0.25 M sucrose, 30 mM Hepes, 5 mM MgCl2 and 1 mM EGTA pH 7.4 supplemented with leupeptin 5 μg/mL and 5 μg/mL antipain, subjected to homogenation for 3 min in a blender followed by 2 min in a bar-mixer, centrifuged 746 ×g 10 min. The supernatant was centrifuged for 70,000 × g for 20 min. The resulting pellet was pottered and washed twice in 0.25 M sucrose, 30 mM Hepes and 5 mM MgCl2 pH 7.4, and resuspended at a concentration of 10 mg protein/mL. All tissue homogenates were stored at − 80 °C until use.

The thioether LHRH analog without yet the modifications at the termini, QHW[AYGA]cRPG was biotechnologically produced by L. lactis, containing the plasmid pIL3BTC, which encodes the thioether bridge introducing enzymes and the transporter, and a second plasmid encoding the LHRH analog fusion peptides. We compared the export efficiency of three constructs: one construct encoding a peptide in which the nisin leader peptide was followed directly by the LHRH analog QHWSYGCRPG (pTP–LHRH1), a second construct encoding a peptide composed of prenisin(1–44) comprising nisin rings A, B and C, fused to QHWSYGCRPG (pLPlhrh, Kluskens et al., 2005), and a third construct encoding prenisin to whose C-terminus the QHWSYGCRPG was fused (pTP–nisin–LHRH1). Qualitative mass spectrometric analyses demonstrated that all three cases led to production of peptide containing thioether-bridged QHW[AYGA]cRPG. Quantitative analyses on SDSPAGE clearly demonstrated that the yield in the case of the second construct was much larger than that of the first and third construct (Fig. 3). Lower production in the case of the first construct which lacks the nisin rings might be caused by the absence of autoinduction (Kuipers, Beerthuyzen, de Ruyter, Luesink & de Vos, 1995), whereas the length of the third construct might cause less efficient export (Rink et al., 2007a). We therefore continued all further experiments with construct 2 and variants thereof, comprising prenisin(1–44). HPLC purification of the trypsin-liberated and terminally modified pEHW[AYGA]cRPG-NH2 peptide produced by L. lactis, displayed a single peak (Fig. 4). Dehydroalanines can spontaneously react with cysteines without the need of NisC. However, mass spectrometry analysis of the Thr variant LHRH peptide obtained from the construct pTP–LHRH1T demonstrated that the Thr was fully dehydrated and CDAP treatment showed that a methyllanthionine ring had formed. Methyllanthionines are not formed spontaneously from dehydrobutyrine and Cys during cultivation and purification and are thus NisC-catalysed (Rink et al., 2007b). Therefore we conclude that the thioether rings formed in the biologically produced LHRH peptides are enzymatically formed. 3.2. pE formation The retention time of the HPLC peaks of all LHRH analogs shifted by 2 min after induction of the conversion of Q to pE. This N-terminal cyclization implies the loss of NH3 and consistently resulted in the mass shift of 17 Da as witnessed by Maldi-TOF mass spectrometry.

2.16. Measurement of peptide breakdown Incubations in a volume of 140 μL were performed in 20 mM NaPO4 pH 5.0 or pH 7.4 at 37 °C, at 0.03, 0.85 and 0.02 μmol peptide/mg tissue homogenate of respectively liver, pancreas or kidney and followed by 5 min incubation at 100 °C to denature proteins. At the applied tissue concentrations complete degradation of natural LHRH occurred within 60 min at pH 7.4, which allowed better evaluation of the differences between natural and thioether stabilized LHRH. pH 7.4 was used to allow cytosolic and extracellular enzymes to work optimally while at pH 5, the lysosomal enzymes are most active. Samples were subsequently put on ice, centrifuged for 10 min at 20,000 x g at 4 °C, after which 95 μL was analysed by HPLC measuring at 280 nm. Peak

Fig. 3. Enhanced production of a hybrid nisin–LHRH construct. The thioether LHRH analog, QHW[AYGA]cRPG was produced by Lactococcus lactis, containing pIL3BTC and a second plasmid, pTP–nisin–LHRH1, encoding a peptide in which the nisin leader peptide was directly fused to QHWSYGCRPG (lane 1), pLPIhrh, encoding a peptide composed of prenisin 1–44 fused to QHWSYGCRPG (lane 2), pTP–nisin–LHRH1, encoding prenisin C-terminally fused to QHWSYGLRPG (lane 3). M is marker lane.

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linear control peptide, QHWSYGLRPG. Hence the thioether bridgeimposed conformational constraint and increased bulkiness did not preclude accessibility and amidating activity of the carboxypeptidase Y. 3.4. Chemical amidation of LHRH

Fig. 4. HPLC separation of thioether LHRH analogs. Continuous line peak LHRH1, thioether LHRH produced by Lactococcus lactis containing pILBTC. Interrupted line peaks: LHRH11 and LHRH12, thioether-bridged isomers or mixtures of isomers obtained by base-assisted sulfur extrusion of 4,7 disulfide bridged LHRH analogs. All analogs have modified termini as in natural LHRH.

We subsequently investigated whether, despite the thioether cyclization, C-terminal amidation would still be possible. We applied an enzymatic and a chemical method for amidation. 3.3. Enzymatic amidation of LHRH Amidation of the C-terminus of an existing peptide is a great challenge. Methods are enzyme-based modification via carboxypeptidase Y (Breddam, Widmer & Meldal, 1991), amidase and most importantly of all PAM (peptidylglycine α-amidating monooxygenase, a vertebrate enzyme) (Eipper, Mains & Glembotski, 1983; Garmendia, Rodriguez, Burell & Villaro, 2002). We here tested enzymatic amidation using carboxypeptidase Y in the presence of an excess of glycinamide. After 10 min the reaction was nearly complete. Prolonged incubation up to 50 min only slowly decreased the level of product. Hence the thioether-bridged peptide is quite stable and protected against prolonged incubation with carboxypeptidase Y. Since carboxypeptidase Y treatment of peptide ending with G yielded only 3% amidated peptide, the enzyme was subsequently tested on QHW[AYGA]cRPA which led to a yield of 36%. Comparable yield was obtained with a

In a first approach we tried to amidate LHRH by making a dehydroalanine extended analog which is cleaved off at low pH yielding an amidated peptide (Chan, Bycroft, Lian & Roberts, 1989). However S11 in various C-terminally extended LHRH analogs was insufficiently dehydrated when produced via Lactococcus containing pIL3BTC. An interesting paper (Nakagawa et al., 1994) reported use of CDAP to cyanylate cysteine residues and subsequently cleave the backbone by using ammonia, which yields an amidated C-terminus. For amidation of the LHRH analog this method requires an additional cysteine. We therefore had the dodecapeptide QHWSYGCRPGCK, which is extended with C11 and K12, modified and produced by L. lactis containing pILBTC. We found that after modification and export out of L. lactis via NisBTC the appropriate peptide QHW[AYGA]cRPGCK was produced. Hence the formed dehydroalanine4 had been coupled to C7 and not to C11. This demonstrated the regiospecific action of NisC. Alkylamide tails which replace amidated G10 have been reported to enhance receptor interaction of linear LHRH (Sealfon et al., 1997; Coy et al., 1975). Therefore the peptide QHWSYGRPCK was treated with CDAP and subsequently with methylamine, ethylamine or propylamine to obtain different alkylamide tails (Fig. 5AB). In the case of methylamine almost 100% conversion to amidated peptide was obtained. Hence this method is clearly more efficient than the amidation using carboxypeptidase Y. The order of amidation efficiency was methylamine N ammonia N ethylamine N propylamine. This chemical amidation was subsequently successfully applied to pE-containing and thioetherbridged LHRH analogs yielding fully modified peptide (Fig. 5AB). Hence the introduction of a thioether ring in LHRH is compatible with the required subsequent modification of the N- and C-terminus. 3.5. Stereoisomers of cyclized LHRH peptides Thioether-bridged LHRH obtained via base-assisted sulfur extrusion (Galande et al., 2003; Fig. 2C) yielded two HPLC peaks (Fig. 4) which we termed LHRH11 and LHRH12. These peptides had identical mass as measured by mass spectrometry and contained thioether rings. Further

Fig. 5. AB. Maldi-TOF spectra of pEHW[AYGA]cRPG-NH2 (A) and pEHW[AYGA]cRP-propylamide (B). Dotted lines correspond to calculated mass spectrum.

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Fig. 6. HPLC-isolated thioether-bridged LHRH variants display identical post source decay mass spectrum and are therefore likely stereoisomers. Thioether-bridged isomers or mixtures of isomers were obtained by base-assisted sulfur extrusion of 4,7 disulfide bridged LHRH analogs. Upper spectrum: Post source decay of HPLC fraction eluting at 21.8 min termed LHRH11, lower spectrum: post source decay of HPLC fraction eluting at 22.6 min, termed LHRH12.

mass spectrometric analyses by post source decay resulted in identical spectra for both peptide fractions (Fig. 6). Therefore the two peaks correspond to at least two different stereoisomers. The biologically produced peptide, termed LHRH1, co-eluted with LHRH11. These data clearly indicate that the presence of a single HPLC peak of the biotechnologically produced peptide results from NisC-mediated stereospecific coupling. All thioether rings in nisin formed by NisC are D,L-lanthionines also called meso-lanthionines (Gross and Morell 1971). Therefore it is likely that also the NisC-introduced thioether ring in LHRH is a D,L-lanthionine ((D-)Ala-S-(L-)Ala). Mass spectrometry analysis can only prove that the biologically produced LHRH1 is chemically identical to the chemically prepared LHRH11 and LHRH12 isomers but does not yield any information about the stereo configuration of the lanthionine. 2D NMR was used to further compare the biological and chemical LHRH variants by looking at the chemical shifts of the backbone protons which are influenced by conformation and stereo configuration. 2D-1H-NMR analysis of LHRH11 and LHRH12 peptide in deuterated DMSO was done by recording 2D-TOCSY and 2D-NOESY spectra. The chemical shift peaks

Table 2 Backbone amide proton chemical shifts of the biologically produced LHRH1 and the chemically prepared LHRH11 and LHRH12 thioether ringed peptides. The backbone amide protons were assigned by recording 2D-TOCSY and 2D-NOESY NMR spectra of the peptides in deuterated DMSO. The chemical shifts are influenced by the D or L configuration of the backbone carbon atom of lanthionine. Residue

H2 W3 A (S) 4 Y5 G6 A (S) 7 R8 G10

LHRH1

LHRH11

LHRH12

Absolute difference

N–H

N–H

N–H

LHRH11/LHRH12

δ (ppm)

δ (ppm)

δ (ppm)

Δδ (ppm)

7.82 8.16 8.30 7.88 8.40 6.80 7.97 7.99

8.06 8.20 8.35 7.83 8.43 6.87 8.07 8.05

7.94 7.79 8.42 8.69 8.60 6.88 8.10 8.02

0.12 0.41 0.07 0.86 0.17 0.01 0.03 0.03

of the backbone amide proton could be assigned in the spectra (Table 2). The differences between the chemical shifts of the backbone amide protons of W3 and Y5 were 0.41 and 0.86 ppm for both LHRH isomers, respectively. This large difference in chemical shift for the residues surrounding lanthionine at position 4 points to a difference in D and L configuration at position 4 between LHRH11 and LHRH12. 2D-TOCSY and 2D-NOESY spectra were also recorded for LHRH1. Despite the low concentration of LHRH1 and a large water peak, the obtained signals with NOESY and TOCSY were sufficient to assign the backbone amide protons. The chemical shifts of the backbone amide protons of LHRH1 were very similar to those recorded for LHRH11, especially for residues 3 to 7 that are part of the ring structure. Only the backbone amide proton of H2 had a deviating and low chemical shift of 7.82 ppm. This might be a result of the relative acidic and watery environment (Bundi and Wuthrich 1979) in which the spectra were recorded. So, LHRH1 is very similar to LHRH11 and not to LHRH12 based on NMR data. Since the biologically produced LHRH1 also co-elutes with LHRH11 it is most likely that both peptides are completely identical and have a D,L-lanthionine configuration. The LHRH12 peptide most likely contains L,L-lanthionine and/or L,D-lanthionine. Although the TOCSY and NOESY spectra only show peaks consistent with enantiopure peptides, it cannot yet be excluded that the lanthionine at position 7 of LHRH shows no discrimination between L or D-configuration in terms of different chemical shift peaks for the backbone amide protons for each configuration. 3.6. Proteolytic resistance We subsequently investigated the proteolytic resistance of the thioether LHRH analogs. The fully modified thioether LHRH analogs and in separate control experiments natural LHRH were exposed to plasma and a variety of protease-rich tissue homogenates and the breakdown was measured by HPLC. Incubations were performed at pH 7.4 (Fig. 7: A1, B1 and C1) and at the lysosomal pH 5.0 (Fig. 7: A2, B2, C2) in the presence of tissue homogenates from pig organs i.e. liver (Fig. 7: A1 A2), pancreas (Fig. 7: B1, B2), kidney cortex (Fig. 7: C1, C2). In plasma in the

R. Rink et al. / Journal of Pharmacological and Toxicological Methods 61 (2010) 210–218

Fig. 7. Proteolytic resistance of thioether-bridged LHRH. The proteolytic resistance of fully modified thioether LHRH11 and LHRH12 isomers (●, ○) and natural LHRH (▲) was measured by HPLC following incubation at pH 7.4 (Fig. A1, B1 and C1) or pH 5.0 (Fig. A2, B2, C2) in the presence of pig homogenates of liver (A1 A2), pancreas (B1, B2) or kidney cortex (C1, C2). For experimental details see the Materials and methods section.

absence of endothelial cells and cell homogenates little if any breakdown at all was observed for both the natural (less than 20% in 2 h) and the thioether-bridged LHRH (less than 12% in 2 h). The results with organ homogenates demonstrate that natural LHRH is degraded more effectively at pH 7.4 than at pH 5.0. This is consistent with the fact that many peptidases are located outside the lysosomes. Natural LHRH which lacks the thioether bridge was in all experiments with organ homogenates more rapidly broken down than the thioether-bridged analogs. Differences between natural LHRH and the thioether-bridged analogs were largest in pancreas homogenate (Fig. 7: B1, B2) which caused degradation of the natural LHRH whereas both the thioetherbridged LHRH analogs were hardly affected. Degradation was faster at pH 7.4 than at pH 5.0. The proteolytic resistance between the thioetherbridged LHRH analogs differed in the liver homogenate and kidney cortex homogenate. The LHRH11 analog, which has identical HPLC retention time as the biologically produced one, LHRH1, was more resistant than LHRH12 (Fig. 7). Likely LHRH1 and LHRH11 are both D,Lthioether-bridged analogs, whereas LHRH12 might perhaps be an L,Land/or L,D-thioether-bridged analog. 4. Discussion The instability of many peptide pharmaceuticals is a major limitation of their therapeutic potential. LHRH is resistant against amino- and carboxypeptidases peptidases by the presence of a pE

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group and by amidation respectively. Like LHRH about 50% of all therapeutic peptides are amidated and several have a pE residue. It is therefore relevant that any stabilization method can be combined with these modifications of the peptide's termini. However, modified peptide termini do not provide protection of cleavage sites within the peptide. Cyclization is a well established way to make peptides resistant against breakdown by peptidases. Chemical cyclization is however cumbersome and costly and usually yields mixtures of isomers which have to be purified. We here exploited L. lactis which contained peptide-modifying enzymes for the production of thioether-bridged peptides. The here presented broadly applicable method involves enzymatic cyclization which is stereo-, regio- and chemospecific. These are important advantages over chemical cyclization methods. The decapeptide hormone, luteinizing hormone release hormone (LHRH: pEHWSYGLRPG-NH2) induces release of luteinizing hormone. LHRH has been subject of much research aiming at the development of potent agonists and antagonists for chemical inhibition of reproduction and antitumor agents (Mezö et al., 2008). Several cyclized analogs, which were obtained by chemical methods, have been reported (Beckers, Bernd, Kutscher & Kuhne, 2001; Bienstock et al., 1993; Rivier, Struthers et al., 2000; Rivier, Porter et al., 2000; Rivier, Jiang et al., 2000; Dutta, Gormly, McLachla & Woodburn, 1989; Mezö et al., 1997). Thioether bridges are more stable than peptide bonds and disulfide bonds (Tugyi, Mezo, Fellinger, Andrea & Hudecz, 2005). Therefore, we examined the possibility to introduce a thioether bridge in LHRH. This peptide's N-terminal pE group and a C-terminal amide are enzymatically introduced in the body where LHRH is endogenously produced. Thioether-bridged LHRH analogs produced by L. lactis lack these modifications of their termini and were therefore introduced after production. In the case of LHRH this amidation is required for functional interaction with the receptor. We here demonstrated that the presence of the thioether bridge in an LHRH analog does not preclude subsequent modifications of the termini either chemically or enzymatically. With respect to enzymatic amidation the presence of a thioether bridge from position 4 to 7 even provided an advantage by blocking the progressive carboxypeptidase action which in principle can completely digest its substrate. Here we demonstrate that a thioether-bridged LHRH analog is significantly more resistant against proteolytic degradation than the natural LHRH in a variety of protease-rich tissue homogenates. Known structures of lantibiotics indicate that the thioether bridges introduced by lantibiotic enzymes are D,L stereoisomers (Gross and Morell, 1971). This suggests that also the enzymatic introduction of thioether rings in non-lantibiotic peptides might be stereospecific, i.e. leading to D,L isomers. D-amino acids by themselves already contribute to proteolytic resistance. The slightly stronger proteolytic resistance of LHRH11 compared to LHRH12 likely results from extra protection by a D-amino acid in position 4 in the case of LHRH11, whereas LHRH12 likely has an L-amino acid in position 4. Consistent with the hypothesis that biologically one D,L isomer will be formed we here obtained only one HPLC peak of thioether LHRH produced by lantibiotic enzyme-containing L. lactis. This biologically produced LHRH analog had the same retention time in HPLC as the most stable isomer, LHRH11, obtained by base-assisted sulfur extrusion. An NMR experiment also pointed to the similarity between LHRH1 and LHRH11, but future work has to establish more detailed conformational features. By contrast thioether-bridged LHRH obtained via baseassisted sulfur extrusion led to two distinct HPLC peaks and NMR measurement indicated two isomers with clear differences in chemical shifts for the backbone amide protons. These data indicate an important advantage of stereospecificity of the microbiological synthesis of thioether-bridged LHRH. We here showed that the introduction of a thioether ring in a peptide hormone analog confers resistance of the peptide against proteolytic degradation. In addition we demonstrated that the

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production of thioether-bridged peptide hormone by L. lactis can be followed by N-terminal pE formation and enzymatic as well as chemical C-terminal amidation. There are several advantages of their biological production, among which are the here demonstrated regiospecific-, and most likely stereospecific, thioether ring formation and the easy generation of libraries of variants as recently demonstrated for nisin (Rink et al., 2007a). The introduction of a thioether ring has also been reported to lead to agonists with enhanced and/or more specific receptor interaction (Rew et al., 2002; Ösapay et al., 1997; Kluskens et al., 2009). The generation of protease-resistant thioether-bridged therapeutic peptides will lead to therapeutic peptides with enhanced therapeutic potential and to analogs that can be delivered orally, the Holy Grail in peptide delivery. Acknowledgement This study was co-financed, without involvement in content of the study, by the European Fund for Regional Development and the Dutch Ministry of Economic Affairs. Ruud M. Scheek and Renee Otten of the NMR facility of the University of Groningen are thanked for their help with NMR and useful discussions. References Ayoub, M., & Scheidegger, D. (2006). Peptide drugs, overcoming the challenges, a growing business. Chemistry Today, 24, 46−48. Beckers, T., Bernd, M., Kutscher, B., & Kuhne, R. (2001). Structure–function studies of linear and cyclized peptide antagonists of the GnRH receptor. Biochemical Biophysical Research Communications, 289, 653−663. Bessalle, R., Kapitkovsky, A., Gorea, A., Shalit, I., & Fridkin, M. (1990). All-D-magainin: Chirality, antimicrobial activity and proteolytic resistance. FEBS Letters, 274, 151−155. Bienstock, R. J., Rizo, J., Koerber, S. C., Rivier, J. E., Hagler, A. T., & Gierasch, L. M. (1993). Conformational analysis of a highly potent dicyclic gonadotropin-releasing hormone antagonists by nuclear magnetic resonance and molecular dynamics. Journal Medicinal Chemistry, 36, 3265−3273. Booth, A. G., & Kenny, A. J. (1974). A rapid method for the preparation of microvilli from rabbit kidney. Biochemical Journal, 142, 575−581. Breddam, K., Widmer, F., & Meldal, M. (1991). Amidation of growth hormone releasing factor(1–29) by serine carboxypeptidase catalysed transpeptidation. International Journal Peptide Protein Research, 37, 153−160. Buist, G., Kok, J., Leenhouts, K. J., Dabrowska, M., Venema, G., & Haandrikman, A. J. (1995). Molecular cloning and nucleotide sequence of the gene encoding the major peptidoglycan hydrolase of Lactococcus lactis, a muramidase needed for cell separation. Journal Bacterioleriology, 177, 1554−1563. Bundi, A., & Wuthrich, K. (1979). 1H-NMR parameters of the common amino acid residues measured in aqueous solutions of the linear tetrapeptides H-Gly-Gly-X-LAla-OH. Biopolymers, 17, 2133−2141. Chan, W. C., Bycroft, B. W., Lian, L. -Y., & Roberts, G. C. K. (1989). Isolation and charactyerization of two degradation products derived from the peptide antibiotic nisin. FEBS Letters, 252, 29−36. Coy, D. H., Vilchez-Martinez, J. A., Coy, E. J., Nishi, N., Arimura, A., & Schally, A. V. (1975). Polyfluoroalkylamine derivatives of luteinizing hormone releasing hormone. Biochemistry, 14, 1848−1851. Dickey, B. F., Fishman, J. B., Fine, R. E., & Navarro, J. (1987). Reconstitution of the rat liver vasopressin receptor coupled to guanine nucleotide-binding proteins. Journal Bioliological Chemistry, 262, 8738−8742. Dutta, A. S., Gormly, J. J., McLachla, P. F., & Woodburn, J. R. (1989). Conformationally restrained cyclic peptides as antagonists of luteinizing hormone-releasing hormone. Biochemical Biophysical Research Communications, 159, 1114−1120. Eipper, B. A., Mains, R. E., & Glembotski, C. C. (1983). Identification in pituitary tissue of a peptide α-amidation activity that acts on glycine-extended peptides and requires molecular oxygen, copper, and ascorbic acid. Proceedings National Academy Sciences USA, 80, 5144−5148. Galande, A. K., Trent, J. O., & Spatola, A. F. (2003). Understanding base-assisted desulfurization using a variety of disulfide-bridged peptides. Biopolymers, 71, 534−551. Garmendia, O., Rodriguez, M. P., Burrell, M. A., & Villaro, A. C. (2002). Immunocytochemical finding of the amidating enzymes in mouse pancreatic A-, B-, and D-cells: A comparison with human and rat. Journal Histochemistry Cytochemistry, 50, 1401−1415.

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