Modulating the Fibrillization of Parathyroid-Hormone (PTH) Peptides: Azo-Switches as Reversible and Catalytic Entities

We here report a novel strategy to control the bioavailability of the fibrillizing parathyroid hormone (PTH)-derived peptides, where the concentration of the bioactive form is controlled by an reversible, photoswitchable peptide. PTH1–84, a human hormone secreted by the parathyroid glands, is important for the maintenance of extracellular fluid calcium and phosphorus homeostasis. Controlling fibrillization of PTH1–84 represents an important approach for in vivo applications, in view of the pharmaceutical applications for this protein. We embed the azobenzene derivate 3-{[(4-aminomethyl)phenyl]diazenyl}benzoic acid (3,4′-AMPB) into the PTH-derived peptide PTH25–37 to generate the artificial peptide AzoPTH25–37 via solid-phase synthesis. AzoPTH25–37 shows excellent photostability (more than 20 h in the dark) and can be reversibly photoswitched between its cis/trans forms. As investigated by ThT-monitored fibrillization assays, the trans-form of AzoPTH25–37 fibrillizes similar to PTH25–37, while the cis-form of AzoPTH25–37 generates only amorphous aggregates. Additionally, cis-AzoPTH25–37 catalytically inhibits the fibrillization of PTH25–37 in ratios of up to one-fifth. The approach reported here is designed to control the concentration of PTH-peptides, where the bioactive form can be catalytically controlled by an added photoswitchable peptide.


Introduction
Fibrillization of proteins and peptides is a supramolecular process [1,2] that leads to the formation of peptide aggregates, containing a cross-β-sheet motif [3]. It involves multiple steps [4] and is associated with many diseases such as Alzheimer's disease, Parkinson's disease or diabetes type II [5][6][7]. However, in the past decades, it has also been associated with amyloids with distinct physiological functions, so-called functional amyloids, which are found in lower organisms [8][9][10][11]. Subsequently, functional amyloids were also discovered in humans, whereby the amyloid can be the active physiological form [12,13] or the storage form of peptide hormones [14].
The parathyroid hormone, abbreviated PTH, is a human hormone secreted by the parathyroid glands [15], with PTH-like peptides also known from other animals [16,17]. It is expressed as a 115 residue pre-pro-protein, whereby the first 25 amino acids at the N-terminus (referred to PTH −31-−7 ) serve as a signaling peptide for the transport to the endoplasmic reticulum and are removed by a signal peptidase [18]. The formed pro-peptide is subsequently transferred to the Golgi apparatus and the N-terminal six amino acids (referred to PTH −6-−1) are proteolytically removed [19]. Before mature PTH 1-84 is released into the blood, it is stored in secretory granules as amyloid fibrils [20]. The physiological role peptide is subsequently transferred to the Golgi apparatus and the N-terminal six acids (referred to PTH−6-−1) are proteolytically removed [19]. Before mature PTH released into the blood, it is stored in secretory granules as amyloid fibrils [20 physiological role is well studied [21,22], being important in the maintenan extracellular fluid calcium and phosphorus homeostasis. The receptor is mainly act through the first 34 N-terminal amino acids [23], wherefore recombinant PTH1recombinant PTH1-34 are approved drugs against osteoporosis, Natpara ® and Fo respectively. However, its fibrillization has barely been investigated. Thus far, it is k that the amyloid fibrils of PTH1-84 are formed by the amino acid residues R25-L37, a thermodynamic stability of the fibrils is sufficiently low to dissociate after dilutio Thus, control over the fibrillization of amyloids and PTH specifically represen important approach for controlling its factual concentration for in vivo applica placing modulators of fibrillization and thus reversible fibrillization into the fo pharmaceutically applicable proteins [24][25][26][27].
In the past decades, the photoinduced switching of protein functionalitie emerged as an important concept to modulate protein function, often by modulati binding specificity between proteins and ligands. Thus, not only enzymes have equipped with photosensitive switches, but also larger protein complexes, involv many physiological or neurological functions [28]. To this end, artificial photoswitch embedded into either the main chain or side chains of polypeptides, in order to c their secondary structures by photoinduced conformational changes o photoswitches. Thereby, a plethora of different photoswitches, such as those based trans-isomerization of azo-dyes [29,30] stilbenes [31] and hemithioindigos [32,33] been developed. Important for the proper use of a specific photoswitch ins polypeptide chain is not only the quest to retain the initial (functional) secondary str of the protein, but also to achieve a reasonably stable conformation after photoswit so as to allow for sufficient time to exert the desired effect. Many examples o sufficiently stable and also reversible photoswitches have been reported, allowing modulate several expects of protein function [34][35][36][37][38][39]. Here, we report on an appro modulate the fibrillization of PTH, equipped with a photoswitch at a specific posit the peptide sequence, in order to reversibly trigger its aggregation/disaggregatio Figure 1).  In view of the functional design of the modified PTH [25][26][27][28][29][30][31][32][33][34][35][36][37] , we sought to embed the photoswitch into a region of the protein where aggregation is still possible, but only in a specific (untriggered) conformation of the photoswitch, whereby fibrillization should be inhibited after the conformational change. As a model system, we chose peptides derived from the PTH fibril core structure, including the amino acids 25R-37L (Figure 1a) [20], which is able to form fibrils itself. In addition, we investigated the influence of both conformations on the fibrillization of the unmodified peptide. As the photoswitch we chose a structural motif from the class of azobenzenes, as they are well known for enabling reversible control of peptide conformation [29,34,[39][40][41]. Specifically we chose the azobenzene derivate 3-{[(4-aminomethyl)phenyl]diazenyl}benzoic acid (3,4 -AMPB; Figure 1b) [42], which is known to introduce a significant geometric change. 3,4 -AMPB displays both: a high photoisomerization yield and a sufficient thermodynamically stability of the cis-isomer [41]. If desired, the photoswitch can be reversed via irradiation at 405 nm, or thermally, with a half-life time of more than 20 h in the dark. We hypothesized that the incorporation of the azobenzene into the backbone would allow us to switch between the cisand the trans-conformation, whereby one of them is able to fibrillize and the other one is not. Furthermore, azobenzenes in their cis-conformation are known to mimic β-hairpins, which allowed us to investigate the hypothesis if the PTH fibrils possess a turn region like amyloid fibrils from other peptides [43][44][45].
ESI-ToF mass spectrometry was performed on a Bruker Daltonics microTOF (Bruker Corporation, Billerica, MA, USA). Samples were dissolved in HPLC-grade solvents (MeOH, THF, or mixtures; Sigma Aldrich, Taufkirchen, Germany) at concentrations of 0.1 mg/mL and measured via direct injection with a flow rate of 180 µL/h using the positive mode with a capillary voltage of 4.5 kV. The spectra were analyzed with otofControl (version 3.4, Bruker Daltonik, Bremen, Germany).

Peptide Synthesis and Purification
Solid-phase peptide synthesis was utilized on an automated peptide synthesizer MultiPep RS (Intavis AG, Koeln, Germany) using standard Fmoc-chemistry and preloaded resins. Standard coupling of all protected natural amino acids was performed as single couplings in dimethylformamid (DMF) using 5 equivalents of amino acids, HCTU as coupling reagents, and 10 equivalents of NMM as base for 1 h at room temperature. Special building groups, such as Fmoc-3,4 -AMPB, were coupled with 3 equivalents using DIC and HOBT in DMF/N-methyl-2-pyrrolidone (NMP) at room temperature and with gentle shaking in the dark overnight.
The N-terminal Fmoc-protecting group was removed by washing the resin with 20% piperidine for 20 min. The final side chain deprotection and cleavage from the resin employed a mixture of trifluoroacetic acid and water (90:10 Vol%) with gentle agitation for 2 h at room temperature.
The crude peptides were purified to >95% purity using preparative RP-HPLC (Gilson, Limburg, Germany). For both analytical and preparative use, the mobile phase was a mixture of water (eluent A) and acetonitrile (eluent B), respectively, each containing 0.1% trifluoroacetic acid. Samples were eluted with a linear gradient from 5% B to 95% B in 15 min for analytical runs and in 90 min for preparative runs on a semipreparative PLRP-S column (300 × 25 mm, 8 µm; Agilent Technologies, Waldbronn, Germany). Finally, all peptides were characterized by analytical HPLC Dionex Ultimate 3000 (Thermo Fisher Scientific, Dreieich, Germany) using a PLRP-S column (150 × 4.6 mm, 3 µm; Agilent Technologies, Waldbronn, Germany) and MALDI-MS (Bruker Microflex LT, Bremen, Germany), which gave the expected [M+H] + mass peaks.

Azobenzene Peptide Photoisomerization
Trans → cis isomerization was performed by irradiating the dissolved peptide in a 1 cm quartz cuvette for 30 min with light of 340 nm wavelength using a 50 W mercury lamp (VEB) and a 340 nm band pass filter (FB340-10, Thorlabs, Bergkirchen, Germany) under stirring. For cis → trans isomerization, the dissolved peptide was irradiated with light of 405 nm wavelength using a 1.4 W LED (M405L4, Thorlabs, Bergkirchen, Germany) for 30 min under stirring.

Transmission Electron Microscopy (TEM)
TEM images were taken with an electron microscope (EM 900; Zeiss, Oberkochen, Germany) at 80 kV acceleration voltage. For preparation, 5 µL of the peptide solution were added on Formvar/Cu grids (mesh 200). After 3 min of incubation, the grids were gently cleaned with water for o1 min and then negatively stained using uranyl acetate (1%, w/v) for 1 min.

Seeding Assay
The seeding assay follows the same procedure as the ThT-monitored fibrillization assay for the determination of the aggregation kinetics. In addition, the final samples contained 20 µM of seeds from trans-AzoPTH 25-37 fibrils. The seeds were prepared via ultrasonification of a 100 µM mature trans-AzoPTH 25-37 fibrils solution (Sonifier W-250 D, Branson Ultraschall, Dietzenbach, Germany; 15 times, 1 s 10% amplitude, 1 s pause).
added on Formvar/Cu grids (mesh 200). After 3 min of incubation, the grids were g cleaned with water for o1 min and then negatively stained using uranyl acetate (1%, for 1 min.

Seeding Assay
The seeding assay follows the same procedure as the ThT-monitored fibrilliz assay for the determination of the aggregation kinetics. In addition, the final sam contained 20 μM of seeds from trans-AzoPTH25-37 fibrils. The seeds were prepare ultrasonification of a 100 μM mature trans-AzoPTH25-37 fibrils solution (Sonifier W-2 Branson Ultraschall, Dietzenbach, Germany; 15 times, 1 s 10% amplitude, 1 s pause)

Chemistry
To investigate the fibrillization behavior of PTH25-37, the azobenzene switch incorporated directly into the peptide backbone. We selected the 3,4′-azobenzene m (Figure 1b) [42]. As it possesses suitable photochemical properties, e.g., an excellent life time with a stability larger than 20 h and switching wavelengths >300 nm. Thes easily addressable by our photophysical equipment and also avoid eve photodegradation. The synthesis was conducted in two steps ( Figure 2a): in the first we conducted the Fmoc-protection of 2 [46], which in the second step reacts in a reaction with an in situ-generated nitroso compound 3 to obtain the Fmoc-protected AMPB 5 in an overall yield of 68%. The modified azobenzene switch 5, bearing the proper functionalities for F chemistry, was incorporated into the peptide backbone of PTH25-37 via solid-phase pe synthesis (Figure 2b). It replaces V31 in the artificial peptide AzoPTH25-37, due to its ce position along the peptide, expecting the largest impact on fibrillization photoswitching. Furthermore, we probed the replacement of D30 or the insertion betw The modified azobenzene switch 5, bearing the proper functionalities for Fmocchemistry, was incorporated into the peptide backbone of PTH 25-37 via solid-phase peptide synthesis (Figure 2b). It replaces V31 in the artificial peptide AzoPTH 25-37 , due to its central position along the peptide, expecting the largest impact on fibrillization after photoswitching. Furthermore, we probed the replacement of D30 or the insertion between D30 and V31, which led to a greater loss of solubility in the fibrillization buffer (240 µM vs. 25 µM vs. 60 µM; Table S1). Thus, several of the generated peptides displayed strongly reduced solubility-an effect that is important for the subsequent investigations. All peptides were obtained in yields of 10-19%, and high purities as proven by both HPLC and MALDI-ToF measurements, in addition to 500 MHz NMR spectroscopy (Figures S1-S5 and S13-S15).

Photophysical Properties
We first studied the photophysical properties of the cis-trans-isomerization of AzoPTH [25][26][27][28][29][30][31][32][33][34][35][36][37] (Figure 1b) by UV/Vis spectroscopy and HPLC analysis in pure water in order to minimize effects of a potential self-assembly and to quantify the generated amounts of the respective cis/trans-modified peptides before and after photoswitching. The UV/Vis spectra for the pure isomers ( Figure S6) were separated from the spectra of trans-enriched AzoPTH 25-37 in the thermodynamically stable state after synthesis and in the cis-enriched photostationary state (PSS, Figure 3) with Wolfram Mathematica 12.2. The trans-isomer displays an absorption maximum at 327 nm (ε = 13,000 cm −1 M −1 ) and a second maximum at 427 nm, while the cis-isomer possesses maxima at 288 nm and 433 nm. Both isomers display two isobestic points at 278 nm and 388 nm. They represent in the thermodynamically stable state a cis-trans ratio of 3:97. Under irradiation with UV light (340 nm), the cis-content could be increased of up to 82% in the cis-enriched PSS. Visible light (405 nm) yields 76% of the transisomer in the trans-enriched PSS via the back reaction. The difference of the trans-content between the trans-enriched PSS at 405 nm and the thermodynamically stable state arises from the overlapping of the n → π* transitions of both isomers at this wavelength [47]. The rate of thermal cis-to-trans isomerization of AzoPTH 25-37 follows first-order kinetics, and was determined by monitoring the increase of the π → π* absorption band at 327 nm ( Figure S7 trans-enriched AzoPTH25-37 in the thermodynamically stable stat the cis-enriched photostationary state (PSS, Figure 3) with Wolfram trans-isomer displays an absorption maximum at 327 nm (ε = 13,00 maximum at 427 nm, while the cis-isomer possesses maxima at 28 isomers display two isobestic points at 278 nm and 388 nm. thermodynamically stable state a cis-trans ratio of 3:97. Under ir (340 nm), the cis-content could be increased of up to 82% in the c light (405 nm) yields 76% of the trans-isomer in the trans-enri reaction. The difference of the trans-content between the trans-enr the thermodynamically stable state arises from the overlapping o of both isomers at this wavelength [47]. The rate of thermal cis-t AzoPTH25-37 follows first-order kinetics, and was determined by of the π → π* absorption band at 327 nm ( Figure S7

Aggregation Kinetics and TEM-Recordings
In order to determine the kinetics of fibril formation of bot isomers a thioflavin T (ThT)-monitored fibrillization assay was co to PTH25-37. ThT is a benzothiazole compound that binds to the cr amyloid fibrils [48]. Causing a large red shift of fluorescence exc turn enables the selective excitation of amyloid fibril-bound ThT observation of fibril formation.
The observations of the ThT-monitored fibrillization assay were supported by negative stain transmission electron microscopy (TEM) after different time points ( Figure  5). After 20 h, amyloid fibrils were only observable for PTH25-37 and trans-AzoPTH25-37 (Figure 5a, 5b), while cis-AzoPTH25-37 formed amorphous aggregates (Figure 5e). Both peptides produced straight fibrils, whereby the single fibrils of PTH25-37 were larger (>6 μm vs. <1.5 μm) and tend to aggregate further. Interestingly, we found fibrils after 60 h for cis-AzoPTH25-37 (Figure 5g), which matched in the morphology those of trans-AzoPTH25-37 even if they were significantly shorter (<300 nm). This may result from the thermal cis-trans-isomerization, as the cis-content decreases and is reduced to 48% after 60 h. In further experiments, we investigated the (catalytic) influence of the AzoPTH25-37 isomers on the fibrillization of PTH25-37 ( Figure 6). We previously observed such catalytic effects of β-turn modified amyloids (Aβ) on the fibrillization of the Alzheimer peptide Aβ1-40 [49]. Thus 100 μM of PTH25-37 were fibrillized in the presence of various concentrations of the respective AzoPTH25-37 isomer (10/20/50/100 μM). Kinetic measurements revealed that the fibrillization behavior of PTH25-37 was affected in the same way as the pure AzoPTH25-37 isomers.
TEM images were recorded for the peptide mixtures after 20 h (Figure 7). In contrast to the pure peptides, we could observe fibrils for all investigated ratios. Interestingly, the fibrils formed by the mixtures exhibit a similar twisted morphology regardless of the used AzoPTH 25-37 isomer. Furthermore, the formation of larger aggregates like for the pure PTH 25-37 ( Figure 5) were only observed for a ratio of 1:10, indicating that the AzoPTH 25-37 inhibits the formation of larger fibril aggregates. TEM images were recorded for the peptide mixtures after 20 h (Fi to the pure peptides, we could observe fibrils for all investigated ratio fibrils formed by the mixtures exhibit a similar twisted morphology reg AzoPTH25-37 isomer. Furthermore, the formation of larger aggregate PTH25-37 ( Figure 5) were only observed for a ratio of 1:10, indicating th inhibits the formation of larger fibril aggregates.

Seeding Experiments
To determine whether both isomers of AzoPTH 25-37 are able to form fibrils or only the trans-isomer, we investigated, if trans-AzoPTH 25-37 fibrils were able to induce seeding [50]. A 100 µM solution of each isomer was treated with 20 µM of mature trans-AzoPTH 25-37 fibrils, and the kinetics of the fibril formation were investigated via a ThT-monitored fibrillization assay (Figure 8). While the fibrillization of the trans-isomer was accelerated compared to the unseeded monomer, we were not able to observe fibrillization for the cis-isomer. This indicates that the cis-isomer is unable to nucleate amyloid formation as well as elongate preformed fibrils. The observed fibrils after 60 h for the cis-isomer are presumably formed by the thermally isomerized trans-isomer. fibrillization assay (Figure 8). While the fibrillization of the trans-isomer wa compared to the unseeded monomer, we were not able to observe fibrillizatio isomer. This indicates that the cis-isomer is unable to nucleate amyloid form as elongate preformed fibrils. The observed fibrils after 60 h for the ci presumably formed by the thermally isomerized trans-isomer.

Conclusions
We here report for the first time a photoswitchable fibrillizing PTH-deri which is able to modulate its fibrillization by embedding an azobenzene ph the middle of PTH25-37. PTH1-84 is a peptide hormone, which is stored a amyloids in secretory granules. Its physiological role is well studied, but detailed information about its exact fibril structure. We used the 3,4′-AMPB to investigate the fibril formation of the fibril core fragment of PTH1-84 by in the azobenzene into the peptide backbone, yielding the modified PTH-der AzoPTH25-37. We could show that the trans-isomer is able to form fibrils, w isomer induces a conformational change that inhibits fibril formation. Hypot can also conclude that there might not be a β-turn in the fibril structure of P cis-conformer would be reminiscent of such a structure, whereas the tran would not. Most importantly, we were able to show that the modified p catalytically inhibit fibrillization of the PTH25-37, underscoring the importanc during this fibrillization process, which in the future allows for a reversible the fibrillization by light as an external stimulus. Studies are in progress to the photocontrol is also possible with the photoswitch at other positions of t and if we can also control the fibrillization of full-length PTH1-84 with o modified peptides. This represents a novel strategy to control bioavailability specifically of PTH peptides and other fibrillating peptides, where n concentration of the bioactive form can be controlled by an added pho peptide, but also the fibrillization as such, important to guide nerve cell rege other directed growth processes in euraryotic cells. For a potential clinical per want to investigate the cytotoxicity of our peptides as well as the ability to i

Conclusions
We here report for the first time a photoswitchable fibrillizing PTH-derived peptide, which is able to modulate its fibrillization by embedding an azobenzene photoswitch in the middle of PTH [25][26][27][28][29][30][31][32][33][34][35][36][37] . PTH 1-84 is a peptide hormone, which is stored as functional amyloids in secretory granules. Its physiological role is well studied, but it still lacks detailed information about its exact fibril structure. We used the 3,4 -AMPB photoswitch to investigate the fibril formation of the fibril core fragment of PTH 1-84 by incorporating the azobenzene into the peptide backbone, yielding the modified PTH-derived peptide AzoPTH [25][26][27][28][29][30][31][32][33][34][35][36][37] . We could show that the trans-isomer is able to form fibrils, while the cisisomer induces a conformational change that inhibits fibril formation. Hypothetically, we can also conclude that there might not be a β-turn in the fibril structure of PTH 1-84 , as the cis-conformer would be reminiscent of such a structure, whereas the trans-conformer would not. Most importantly, we were able to show that the modified peptides can catalytically inhibit fibrillization of the PTH 25-37 , underscoring the importance of seeding during this fibrillization process, which in the future allows for a reversible triggering of the fibrillization by light as an external stimulus. Studies are in progress to investigate if the photocontrol is also possible with the photoswitch at other positions of the backbone and if we can also control the fibrillization of full-length PTH 1-84 with ours or other modified peptides. This represents a novel strategy to control bioavailability of proteins, specifically of PTH peptides and other fibrillating peptides, where not only the concentration of the bioactive form can be controlled by an added photoswitchable peptide, but also the fibrillization as such, important to guide nerve cell regeneration and other directed growth processes in euraryotic cells. For a potential clinical perspective, we want to investigate the cytotoxicity of our peptides as well as the ability to influence the fibrillization of larger PTH-derived peptides (e.g., PTH 1-34 and PTH 1-84 ) in vitro and in vivo. As known from other azobenzene containing drugs/prodrugs (e.g., Prontosil), the azobenzene moiety is metabolized in liver tissue via azoreductases, yielding two aniline moieties or through intestinal microbes [51,52]. This is potentially important for the photoswitching inside cells by light, allowing them to tune the reversible fibrillization of other amyloidogenic peptides, which important for regeneration of nerve cells, as reported earlier. Thus, peptide fibrils can seed potential harmful amyloidogenic peptides, which is known from recent work quite prominently [53]. This is a strategy to trigger fiber-formation from the outside via photochemical triggering-thus avoiding the toxic effects of the fibers outside the cells but enabling triggered fibrillization inside the cell to exert the desired effects, allowing them to promote the recovery of spinal cord injuries.