Esterification of PQQ Enhances Blood-Brain Barrier Permeability and

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Letter

Esterification of PQQ enhances blood-brain barrier permeability and inhibitory activity against amyloidogenic protein fibril formation Kaori Tsukakoshi, Wataru Yoshida, Masaki Kobayashi, Natsuki Kobayashi, Jihoon Kim, Toshisuke Kaku, Toshitsugu Iguchi, Kazuo Nagasawa, Ryutaro Asano, Kazunori Ikebukuro, and Koji Sode ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00355 • Publication Date (Web): 03 Aug 2018 Downloaded from http://pubs.acs.org on August 5, 2018

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ACS Chemical Neuroscience

Abstract Several neurodegenerative diseases have a common pathophysiology where selective damage to neurons results from the accumulation of amyloid oligomer proteins formed via fibrilization. Considering that the formation of amyloid oligomers leads to cytotoxicity, the development of chemical compounds that are able to effectively cross the blood-brain barrier (BBB) and inhibit this conversion to oligomers and/or fibrils is essential for neurodegenerative disease therapy. We previously reported that pyrroloquinoline quinone (PQQ) prevented aggregation and fibrillation of α-synuclein, amyloid β1–42 (Aβ1–42), and mouse prion protein. To develop a novel drug against neurodegenerative diseases based on PQQ, it is necessary to improve the insufficient BBB permeability of PQQ. Here, we show that an esterified compound of PQQ, PQQ-trimethylester (PQQ-TME), has twice the BBB permeability than PQQ in vitro. Moreover, PQQ-TME exhibited greater inhibitory activity against fibrillation of α-synuclein, Aβ1–42, and prion protein. These results indicated that esterification of PQQ could be a useful approach in developing a novel PQQ-based amyloid inhibitor.

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Introduction Neurodegenerative diseases are characterized by the formation and accumulation of misfolded proteins such as α-synuclein, amyloid β (Aβ), and prion protein that are also implicated in the pathogenesis of Parkinson’s disease, Alzheimer’s disease, and prion disease, respectively.1-5 The causative proteins are able to transform into a β-strand-rich conformation, acquire oligomeric status, and subsequently form supramolecular assemblies and amyloids. It has been hypothesized that these oligomers can be cytotoxic; therefore, inhibitors of the initial protein conformational change may allow a therapeutic approach to these neurodegenerative diseases.6-17 We previously reported that pyrroloquinoline quinone (PQQ) prevented fibril formation of α-synuclein, Aβ1-42, and mouse prion protein in vitro.18-19 Further cytotoxicity analysis suggested that PQQ inhibited the formation of cytotoxic oligomers of α-synuclein and Aβ1-42 in vitro.20 We also found that PQQ-modified α-synuclein36–46 peptide, which is a conjugate of PQQ and a partial sequence of α-synuclein, prevented α-synuclein amyloid fibril formation, but did not inhibit Aβ1–42 fibril formation.21-22 These results indicated that PQQ would be a leading compound in designing amyloid inhibitors. Adequate drug delivery into the central nervous system is essential when developing a therapeutic agent for neurodegenerative diseases, since the blood-brain barrier (BBB) acts as a physical barricade that prevents drug penetration into the brain.23-24 Low molecular weight, lack of ionization, and lipophilicity are all considered favorable factors for BBB penetration. The chemical structure and water solubility of PQQ suggests that it would not easily cross the BBB. In particular, oral administration of PQQ showed low drug-brain penetration in mouse models.25 This suggests that it is necessary to improve the BBB permeability of PQQ for the development of superior PQQ-based amyloid inhibitors. Several inhibitors of fibril formation have a quinone structure that covalently binds to the lysine residues of the amyloid-forming protein via a Schiff-base formation to prevent fibrillation.7,17 PQQ also has a quinone structure that would be important for the inhibition of amyloid-forming protein fibrillation. Therefore, we focused on the esterified compound of PQQ, PQQ-trimethylester (PQQ-TME), which has a lower water solubility than PQQ but contains a quinone structure (Figure 1).26 In this study,


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we analyzed inhibitory activity of PQQ-TME against fibrillation of α-synuclein, Aβ1–42, and mouse prion protein, and the BBB permeability in vitro. Results and Discussion Synthesis of PQQ-TME and effect of PQQ-TME on cell viability PQQ, initially discovered from microorganism and foods,27 is currently commercially available for use in dietary supplements.28 As introduced by Nakano et al.,29 no obvious toxic effects of PQQ were confirmed in in vitro tests and in vivo assays utilizing mice and rats. Therefore, we expected that PQQ might be a good lead compound for anti-amyloidosis drugs on the basis of its inhibition of amyloid-formation. Since the 1990s, PQQ derivatives have been developed for the purpose of not only health-promoting benefits30-32 but also for evaluation of the chemical functions of PQQ as an enzyme

cofactor.33

Considering the BBB permeability, we decided to synthesize and test PQQ-TME as an amyloid inhibitor. It was reported that PQQ-TME was chemically synthesized and an injection test of several doses of PQQ-TME (0.1~1.0 mg/kg) in rats has been performed,31 suggesting low toxicity of the PQQ-TME as well as PQQ. The 1H NMR spectral data of synthetic PQQ-TME were identical to those reported by Urakami et al.26 Finally, PQQ-TME was obtained as a red powder with 57% yield. Considering an application of PQQ and PQQ-TME for BBB testing, consisting of a primary culture and future in vivo use, we investigated the effect of PQQ and PQQ-TME on neuronal cell viability. As described previously, safety of PQQ was well-defined and PQQ can be classified as an ingredient suitable for dietary supplements. Because PQQ-TME was tested using rats for secretion of nerve growth factor, we conducted a cell viability analysis using human neuroblastoma SH-SY5Y cells with PQQ and PQQ-TME added to the cell culture medium (Figure 2). In this assay, we measured the absorbance of formazan, which results from bioreduction of MTS tetrazolium compound by dehydrogenase enzymes in living cells. Up to 200 µM PQQ prompted no decrease in absorbance (Figure 2), indicating that PQQ was non-toxic to SH-SY5Y cells. Unfortunately, the absorbance was greatly reduced in SH-SY5Y cells treated with 200 µM of PQQ-TME for 25 h. Since we did not find absorbance change in SH-SY5Y cells treated with solvent control containing DMSO 4 ACS Paragon Plus Environment


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(data not shown), this marked decrease in absorbance might be caused by PQQ-TME. Thus, it should be noted that long-term use of PQQ-TME at high concentrations may lead to neural cell dysfunction. However, doses up to 100 µM PQQ-TME showed no toxicity toward SH-SY5Y cells. PQQ-TME prevents fibrillation of α-synuclein To evaluate the inhibitory activity of PQQ-TME on amyloid formation of α-synuclein, a thioflavin T (TfT) assay was performed. When 70 µM of α-synuclein was incubated, the fluorescence intensity of TfT increased after 20 h of incubation, indicating that α-synuclein formed amyloid fibril structures (Figure 3A). With the addition of PQQ-TME, amyloid fibril formation was prevented (Figure 3A). After a 98-h incubation, the fluorescence intensity decreased to 40%, 20%, and 5% with the addition of 5, 20, and 50 µM of PQQ-TME, respectively (Figure 3B, black circles). When α-synuclein was incubated with 5, 20, and 50 µM of PQQ for 98 h, the fluorescence intensity decreased to 60%, 50%, and 20%, respectively (Figure 3B, white diamonds). To analyze the total aggregation of α-synuclein, a light scattering assay that detects large particles such as fibrillar and amorphous proteins was performed. The light scattering at 500 nm for the 122-h-incubated α-synuclein sample with both PQQ and PQQ-TME was measured. The light scattering at 500 nm for α-synuclein was decreased with the addition of PQQ-TME, and the inhibitory effect was greater than that of PQQ (Figure 3C). These results indicated that PQQ-TME prevents fibrillation of α-synuclein and has a higher inhibitory activity than PQQ. We previously reported that PQQ binds to α-synuclein and that the resulting PQQ-conjugated α-synuclein inhibits fibrillation of α-synuclein. To investigate whether PQQ-TME also forms conjugates, we analyzed the spectra of α-synuclein after incubation with PQQ-TME over 170 h (Figure 3D). PQQ-TME showed absorbance at around 340 nm and 280 nm. Although α-synuclein did not show any particular absorbance other than at 280 nm, α-synuclein incubated with PQQ-TME showed a different spectrum from that of α-synuclein, suggesting that the incubation of α-synuclein with PQQ-TME resulted in the formation of PQQ-TME-conjugated α-synuclein. PQQ-TME prevents fibrillation of amyloid β1–42, and mouse prion protein 5 ACS Paragon Plus Environment

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To investigate whether PQQ-TME prevents fibrillation of Aβ1-42, a thioflavin T (TfT) assay were performed. When 25 µM of Aβ1-42 was incubated, the fluorescence intensity of TfT increased after 1 h and was saturated after 4 h (Figure 4A). On the other hand, PQQ-TME dose-dependently prevented the amyloid fibril formation of Aβ1-42. Treatment with 50 µM PQQ-TME did not change the fibrilization kinetics of Aβ1-42; however, the fluorescence intensity decreased to 70% and 55% with the addition of 150 and 250 µM PQQ-TME, respectively. The addition of 150 or 250 µM PQQ resulted in the formation of 92% and 87% of the fibrils formed, indicating that PQQ-TME had a higher inhibitory activity than PQQ against Aβ1-42 fibril formation (Figure 4B). Next, the inhibitory activity of PQQ-TME against mouse prion protein fibril formation was analyzed. To induce the fibril formation of prion protein, a cell-free conversion system was utilized and fibril formation was monitored by TfT. When 5 µM prion protein was incubated, the fluorescence intensity of TfT was increased and fibril formation was dose-dependently prevented by addition of PQQ-TME (Figure 4C). After a 65-h incubation, the fluorescence intensity decreased to 35% and 15% with the addition of 50 and 100 µM PQQ-TME, respectively, although an 85% and 35% inhibition was also observed with the addition of 50 and 100 µM PQQ (Figure 4D). These results showed that the inhibitory activities of PQQ-TME against fibril formation of α-synuclein, Aβ1-42, and mouse prion protein, were different. We were not able to observe potent inhibition of Aβ1-42 fibrilization by PQQ-TME, in particular, with less than 150 µM of PQQ-TME. This may result from the fact that the quinone structure of PQQ-TME, as well as PQQ, prevents fibrillation by covalently binding to the lysine residues of the amyloid-forming protein via a Schiff-base formation, which correlates to the number of lysine residues in the amyloid protein.18 In brief, α-synuclein contains 15 lysine residues, Aβ1-42 contains only two lysine residues, and mouse prion protein contains 11 lysine residues. According to the solved fibril structures of Aβ1-4234 and α-synuclein35, the lysine residues are located or partially located at β-strands in parallel cross-β structure, respectively. Therefore, as we described previously, direct binding of the compound to the lysine residues may be associated with the amyloid fibrilization inhibiting effect of PQQ and PQQ-TME. Given that high concentrations of PQQ-TME might be connected to cellular dysfunction, a methodology would be necessary to facilitate the conjugation of PQQ-TME to amyloidogenic proteins having a low number of lysine residues, such as Aβ1-42. We also demonstrated that PQQ-TME exhibited 6 ACS Paragon Plus Environment


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higher inhibitory activity than PQQ when added to Aβ1-42, and mouse prion protein, which suggested that intermolecular hydrophobic interactions between amyloid proteins are important for amyloid fibril formation. Thus, the increased hydrophobicity may be a key factor in enhancing the reaction of PQQ-TME with amyloid proteins. PQQ-TME has BBB permeability activity than PQQ The BBB permeability of PQQ and PQQ-TME was measured by means of in vitro BBB model (Figure 5). Sodium fluorescein and BSA were utilized as a positive and negative control, respectively. PQQ has similar BBB permeability to sodium fluorescein, which has been widely used as a tracer in studies on BBB permeability.36 On the other hand, PQQ-TME showed two times greater BBB permeability than that of PQQ. These results indicated that esterification of PQQ enhances BBB permeability. Little is known about the BBB permeability of amyloid inhibitory compounds, whereas curcumin showed a low BBB permeability, so utilizing delivery by nanoparticles was considered.37 A recent paper from Pervin et al.38 reported that polyphenol (–)-epigallochatechin gallate (EGCG) penetrated the in vitro BBB model with permeability of 2.8% in 30 min. Therefore, to reveal effective administration of amyloid inhibitors, low BBB permeability of the compounds still remains as an unsolved issue. In this study, we demonstrated esterification of PQQ was a useful modification for increasing BBB permeability, and we discovered chemically-modified PQQ derivatives have the potential for development of new amyloid inhibitors. In summary, we have found that PQQ-TME prevented fibrillation of α-synuclein, Aβ1–42, and mouse prion protein in vitro and had a greater inhibitory activity than PQQ. The inhibitory activity might depend on the number of lysine residues in the amyloid protein, suggesting that the quinone structure of PQQ-TME has an important role in the inhibition of fibril formation by enhancing the reaction of PQQ-TME and amyloid proteins. Additionally, PQQ-TME is more hydrophobic than PQQ and demonstrates a twofold greater BBB permeability. These results demonstrate that the esterification of PQQ enhances its inhibitory activity against amyloid protein fibrillation and BBB permeability, thus making it a novel option for future neurodegenerative disease therapy.


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Methods Chemicals Thioflavin T (TfT) was purchased from Sigma. Amyloid β protein (human, 1-42) (Trifluoroacetate form) was a product of Peptide Institute, Inc. PQQ was kindly donated by Mitsubishi Gas Chemical. Preparation of α-synuclein and mouse prion protein Human wild-type α-synuclein was expressed in the E. coli BL21 (DE3) transfected with the pET28a(+)/α-synuclein plasmid and purified, as described previously.19 Mouse prion protein was expressed in the E. coli Rosetta transfected with the pET22/prion protein plasmid and purified as described previously.18 Synthesis of PQQ-trimethylester Synthesis of PQQ-TME was carried out according to Urakami et al..26 Briefly, a solution of PQQ in dimethyl formamide (DMF) with anhydrous potassium carbonate and dimethyl sulfate was stirred under N2 at 25°C. The reaction was stopped by addition of hydrochloric acid, and the products were extracted with chloroform. The organic layer was washed with brine, dried over magnesium sulfate (MgSO4), and concentrated in vacuo. The resulting residue was purified by silica gel chromatography (ethyl acetate) to afford PQQ-TME as an orange powder. The synthesized PQQ-TME was confirmed by 1H NMR (JNM-AL 300, JEOL). Amyloid fibril formation analysis Purified α-synuclein was ultracentrifuged (150,000 g, 1 h, 4°C) to remove any aggregates. PQQ-TME was mixed with 1.0 mg/mL (70 µM) α-synuclein in PBS buffer containing 0.02% NaN3. For each sample, 250 µL was aliquoted in triplicate into a 96-well microtiter plate together with a Teflon ball and incubated at 37°C with shaking at 700 rpm. Amyloid formation was monitored by the TfT assay. Aliquots of 2.5 µL were removed from the incubated sample and added to 250 µL of 25 µM TfT in PBS buffer. TfT fluorescence was recorded at 486 nm with excitation at 450 nm using an ARVO MX 1420 multilabel counter (PerkinElmer, Waltham, MA, USA). Light scattering at 500 nm was used to monitor the total aggregation of these samples incubated for 122 h. 8 ACS Paragon Plus Environment


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For analysis of Aβ1–42 fibril formation, Aβ1–42 peptide was dissolved in DMSO to make a 3.8 mg/mL stock solution. The stock solution was centrifuged at 250,000g, 4°C for 3h to remove any aggregates. PQQ-TME was mixed with 25 µM Aβ1–42 diluted in PBS buffer with 0.02% NaN3 and then incubated at 37°C in a 0.5 mL tube. Aliquots of 10 µL were removed from the incubated sample and TfT fluorescence was recorded as described above. For analysis of prion protein fibril formation, a cell-free conversion system was used as previously reported by Bocharova et al.39 Lyophilized mouse prion was solubilized in 7.5 M guanidine hydrochloride (GdnHCl). This solution (200 µL) was adjusted to 30 µM of prion protein and 0, 60, 300, 600 µM of PQQ or PQQ-TME in 6 M GdnHCl. The samples were preincubated for 24 h at room temperature. After incubation, 1 mL of conversion buffer (3.6 M Urea, 50 mM phosphate buffer (P.P.B.), 150 mM NaCl, pH 6.8) was added. The samples containing 5 µM prion protein were incubated in 1.2 mL of 1 M GdnHCl, 3 M urea, 150 mM NaCl, 1% DMSO, 50 mM P.P.B. (pH 6.8) at 37°C with shaking at 900 rpm. The samples collected during the incubation time course were diluted into 5 mM sodium acetate buffer (pH 5.5) to a final concentration of prion protein of 0.6 µM, and then TfT was added to a final concentration of 10 µM. TfT fluorescence was recorded as described above. Measurement of UV-Vis spectra of PQQ-TME-conjugated α-synuclein PQQ-TME-conjugated α-synuclein was prepared by incubating 105 µM α-synuclein with 200 µM PQQ-TME at 37°C for 100 h. The incubated sample was loaded onto a NAP-5 column (GE Healthcare) to remove the intact PQQ-TME. The eluted fraction was collected as PQQ-TME-conjugated α-synuclein, and 35 µM PQQ-TME-conjugated α-synuclein was used for measurement of UV-Vis spectra (250 to 600 nm). Cytotoxicity assay A

3-(4,5-dimethylthiazole-

2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium inner salt (MTS) assay kit (CellTiter 96 AQueous nonradioactive cell proliferation assay; Promega, Madison, WI) was utilized to investigate any effect of PQQ-TME on cell viability. Human neuroblastoma SH-SY5Y cells (10,000 cells in 100 µL of DMEM/F12 medium containing 10% fetal bovine serum, penicillin-streptomycin glutamine (Gibco), and 9 ACS Paragon Plus Environment

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minimum Eagle's medium nonessential amino acid solution (Gibco)) were plated on 96-well, collagen-coated, flat-bottomed plates (Corning Incorporated). The cells were incubated overnight to allow the cells to adhere to the well. Ten mM of PQQ or PQQ-TME dissolved in 100% DMSO was diluted to 6.3, 13, 25, 50, 100, 200 µM with the culture medium, and solvent (DMSO) control was also prepared in the same manner as the dilution of PQQ and PQQ-TME. After removal of the culture medium by aspiration, a 100 µL aliquot of diluted-PQQ, PQQ-TME, solvent control, or the culture medium was added to each well. The cells were incubated for 25 h at 37°C and then each well was washed three times with PBS. After removal of the final wash buffer, detection reagent was added and the cells were incubated for 15 h at 37°C. After incubation, we measured absorbance at 490 nm (signal) derived from the formazan product and 655 nm (reference) derived from nonspecific absorbance. The absorbance recorded at a wavelength of 655 nm was subtracted from the absorbance at 490 nm when analyzing the absorbance change. We calculated percentage of cell viability with 100% representing the cells incubated in culture medium.

BBB permeability assay BBB permeability of PQQ and PQQ-TME was analyzed by BBB KitTM PET-12 (PharmaCo-Cell, Co. Ltd., Nagasaki, Japan). PQQ, PQQ-TME, sodium fluorescein (Na-F) and bovine serum albumin (BSA) were dissolved in DPBS-H buffer (10 mM HEPES, 8.1 mM Na2HPO4, 1.47 mM KH2PO4, 0.9 mM CaCl2, 0.49 mM MgCl2, 2.68 mM KCl, 137 mM NaCl, 25 mM d-glucose, pH7.3) with 0.2% DMSO and then 500 µL of sample was added to Transwell clear inserts of the kit (200 µM PQQ, 35 µM PQQ-TME, 200 µM Na-F, and 610 µM BSA). After 20 min incubation, the concentration of the analytes that cross the insert was determined by measuring the intrinsic fluorescence intensity (370 nm excitation and 486 nm emission for PQQ; 375 nm excitation and 453 nm emission for PQQ-TME; 485 nm excitation and 530 nm emission for Na-F). The BSA concentration was determined by a DC protein assay kit (Bio-Rad). The Papp (cm/s) value indicating in vivo permeability of drugs was calculated according the manufacturer’s instructions.



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Author Information Corresponding Author *Mailing address: Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC27599, USA. E-mail: [email protected] Author Contributions K.S. designed and planned the overall study. M.K., T.I., and K.N. synthesized PQQ-TME. M.K., N.K., and J.K. prepared amyloidogenic proteins and performed biochemical characterization studies. K.T., T.K., and R.A. performed the cellular toxicity study. K.T. and M.K. analyzed the data. K.T., W.Y., K.I. and K.S. wrote the manuscript and contributed to figure preparation. Conflict of Interest The authors declare that they have no conflicts of interests. Funding Sources This work was supported by JSPS KAKENHI Grant Number JP20360369.

Acknowledgement The authors thank Dr. Takashi Nonaka (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan) for kindly providing SH-SY5Y cells and advising cell culture techniques.

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Figure legend Figure 1. Structure of PQQ (left) and PQQ-TME (right). Figure 2. Cell viability assay using human neuroblastoma SH-SY5Y cells treated with PQQ (left, black bars) and PQQ-TME (right, gray bars). All experiments were performed in triplicate. Figure 3. Effect of PQQ-TME on the amyloid fibril formation of α-synuclein. (A) The time course of α-synuclein fibril formation, as determined by TfT fluorescence assay analysis. No additive (70 µM α-synuclein) (black); +5 µM PQQ-TME (blue); +20 µM PQQ-TME (pink); +50 µM PQQ-TME (red). (B) The TfT fluorescence intensity after 98 h incubation of α-synuclein with PQQ or PQQ-TME. PQQ (white diamonds); PQQ-TME (black circles). (C) Light scattering analysis of α-synuclein incubated with PQQ or PQQ-TME. Lane 1: No additive (70 µM α-synuclein); lane 2: + 5 µM PQQ; lane 3: + 20 µM PQQ; lane 4: + 50 µM PQQ; lane 5: + 5 µM PQQ-TME; lane 6: + 20 µM PQQ-TME; lane 7: + 50 µM PQQ-TME. (D) UV-Vis spectra of α-synuclein, PQQ-TME, and PQQ-TME-conjugated α-synuclein: α-synuclein (black), PQQ-TME (blue), PQQ-TME-conjugated α-synuclein (red). Figure 4. Effect of PQQ-TME on the amyloid fibril formation of Aβ1–42, and mouse prion protein. (A) The time course of Aβ1–42 fibril formation, as determined by TfT fluorescence assay analysis. No additive (25 µM Aβ1–42) (black); +50 µM PQQ-TME (blue); +150 µM PQQ-TME (pink); +250 µM PQQ-TME (red). (B) The TfT fluorescence intensity after 12 h incubation of Aβ1–42 with PQQ or PQQ-TME. PQQ (white diamonds); PQQ-TME (black circles). (C) The time course of prion protein fibril formation, as determined by TfT fluorescence assay analysis. No additive (5 µM prion protein) (black); +10 µM PQQ-TME (blue); +50 µM PQQ-TME (pink); +100 µM PQQ-TME (red). (D) The TfT fluorescence intensity after 43 h incubation of prion protein with PQQ or PQQ-TME. PQQ (white diamonds); PQQ-TME (black circles). Figure 5. The BBB permeability of PQQ and PQQ-TME analyzed by in vitro BBB model (n=1).



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Page 17 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Structure of PQQ (left) and PQQ-TME (right). 711x325mm (96 x 96 DPI)

ACS Paragon Plus Environment


Cell viability assay using human neuroblastoma SH-SY5Y cells treated with PQQ (left, black bars) and PQQTME (right, gray bars). All experiments were performed in triplicate. 67x47mm (300 x 300 DPI)


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Page 19 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Effect of PQQ-TME on the amyloid fibril formation of α-synuclein. (A) The time course of α-synuclein fibril formation, as determined by TfT fluorescence assay analysis. No additive (70 µM α-synuclein) (black); +5 µM PQQ-TME (blue); +20 µM PQQ-TME (pink); +50 µM PQQ-TME (red). (B) The TfT fluorescence intensity after 98 h incubation of α-synuclein with PQQ or PQQ-TME. PQQ (white diamonds); PQQ-TME (black circles). (C) Light scattering analysis of α-synuclein incubated with PQQ or PQQ-TME. Lane 1: No additive (70 µM αsynuclein); lane 2: + 5 µM PQQ; lane 3: + 20 µM PQQ; lane 4: + 50 µM PQQ; lane 5: + 5 µM PQQ-TME; lane 6: + 20 µM PQQ-TME; lane 7: + 50 µM PQQ-TME. (D) UV-Vis spectra of α-synuclein, PQQ-TME, and PQQ-TME-conjugated α-synuclein: α-synuclein (black), PQQ-TME (blue), PQQ-TME-conjugated α-synuclein (red). 1143x857mm (68 x 68 DPI)



Effect of PQQ-TME on the amyloid fibril formation of Aβ1–42, and mouse prion protein. (A) The time course of Aβ1–42 fibril formation, as determined by TfT fluorescence assay analysis. No additive (25 µM Aβ1–42) (black); +50 µM PQQ-TME (blue); +150 µM PQQ-TME (pink); +250 µM PQQ-TME (red). (B) The TfT fluorescence intensity after 12 h incubation of Aβ1–42 with PQQ or PQQ-TME. PQQ (white diamonds); PQQTME (black circles). (C) The time course of prion protein fibril formation, as determined by TfT fluorescence assay analysis. No additive (5 µM prion protein) (black); +10 µM PQQ-TME (blue); +50 µM PQQ-TME (pink); +100 µM PQQ-TME (red). (D) The TfT fluorescence intensity after 43 h incubation of prion protein with PQQ or PQQ-TME. PQQ (white diamonds); PQQ-TME (black circles). 1143x857mm (68 x 68 DPI)


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The BBB permeability of PQQ and PQQ-TME analyzed by in vitro BBB model (n=1). 467x606mm (96 x 96 DPI)


Esterification of PQQ Enhances Blood-Brain Barrier Permeability and

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